Imbernon Tanycytes Control Hyphotalamic Liranglutide Utake and Its Anti Obesity Actions

Short Article
Tanycytes control hypothalamic liraglutide uptake

and its anti-obesity actions
Graphical abstract Authors
Monica Imbernon, Chiara Saponaro,
Hans Christian Cederberg Helms, ...,
Serge Luquet, Caroline Bonner,
Vincent Prevot
Correspondence
caroline.bonner@univ-lille.fr (C.B.),
vincent.prevot@inserm.fr (V.P.)
In brief
Liraglutide, a drug approved to treat
obesity and type 2 diabetes, must enter
the brain to exert its effects. Imbernon
and Saponaro et al. report that tanycytes,
which are specialized ependymoglial
cells, express GLP1R and transport
liraglutide into the mediobasal
hypothalamus, effectively reducing food
intake, increasing fatty acid oxidation,
and inducing weight loss.
Highlights
d Liraglutide enters the brain through tanycytes of the
mediobasal hypothalamus
d Endothelial cells of the blood-brain barrier do not

express GLP1R
d Tanycytic liraglutide transport is essential to activate target

hypothalamic neurons
d Blocking tanycytic transport or GLP1R impairs the anti-

obesity actions of liraglutide
Imbernon et al., 2022, Cell Metabolism 34, 1054–1063

July 5, 2022 ª 2022 Elsevier Inc.
https://doi.org/10.1016/j.cmet.2022.06.002 ll
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Tanycytes control hypothalamic liraglutide
uptake and its anti-obesity actions
Monica Imbernon,1,7 Chiara Saponaro,2,7 Hans Christian Cederberg Helms,1,3 Manon Duquenne,1 Daniela Fernandois,1
Eleonora Deligia,1 Raphael G.P. Denis,4 Daniela Herrera Moro Chao,4 Sowmyalakshmi Rasika,1 Bart Staels,5
François Pattou,2 Frank W. Pfrieger,6 Birger Brodin,3 Serge Luquet,4 Caroline Bonner,2,7,* and Vincent Prevot1,7,8,*
1Univ. Lille, Inserm, CHU Lille, Laboratory of Development and Plasticity of the Neuroendocrine Brain, Lille Neuroscience & Cognition, UMR-S
1172, European Genomic Institute for Diabetes (EGID), 59000 Lille, France
2Univ. Lille, CHU Lille, Inserm U1190, EGID, Institut Pasteur de Lille, 59000 Lille, France
3Department of Pharmacy, University of Copenhagen, Copenhagen 2100, Denmark
4Université de Paris, BFA, UMR 8251, CNRS, 75013 Paris, France
5Univ. Lille, Inserm, CHU Lille, Institut Pasteur de Lille, U1011-EGID, Lille, France
6Centre National de la Recherche Scientifique, Université de Strasbourg, Institut des Neurosciences Cellulaires et Intégratives, Strasbourg,
France
7These authors contributed equally
8Lead contact
*Correspondence: caroline.bonner@univ-lille.fr (C.B.), vincent.prevot@inserm.fr (V.P.)

https://doi.org/10.1016/j.cmet.2022.06.002
SUMMARY
Liraglutide, an anti-diabetic drug and agonist of the glucagon-like peptide one receptor (GLP1R), has recently
been approved to treat obesity in individuals with or without type 2 diabetes. Despite its extensive metabolic
benefits, the mechanism and site of action of liraglutide remain unclear. Here, we demonstrate that liraglutide
is shuttled to target cells in the mouse hypothalamus by specialized ependymoglial cells called tanycytes,
bypassing the blood-brain barrier. Selectively silencing GLP1R in tanycytes or inhibiting tanycytic transcyto-
sis by botulinum neurotoxin expression not only hampers liraglutide transport into the brain and its activation
of target hypothalamic neurons, but also blocks its anti-obesity effects on food intake, body weight and fat
mass, and fatty acid oxidation. Collectively, these striking data indicate that the liraglutide-induced activation
of hypothalamic neurons and its downstream metabolic effects are mediated by its tanycytic transport into
the mediobasal hypothalamus, strengthening the notion of tanycytes as key regulators of metabolic homeo-
stasis.
INTRODUCTION carries an additional albumin-binding fatty-acid side chain

(Garber, 2011) that reduces its cleavage by DPP-4 and prolongs
Glucagon-like peptide 1 (GLP1; 7–36 amide), an incretin hor- its half-life when compared to endogenous GLP1 (Kim and Egan,
mone mainly secreted by intestinal L-cells in response to 2008). Among its various metabolic effects, liraglutide reduces
glucose and other ingested nutrients, induces insulin secretion food intake and body weight, leading to its recent approval as
via its receptor, GLP1R, in a glucose-regulated manner (Kjems an anti-obesity treatment for individuals with or without T2D
et al., 2003; Peyot et al., 2009). GLP1 is also endogenously pro- (Drucker et al., 2017).
duced in several metabolic organs including pancreatic alpha Extensive studies have shown that liraglutide regulates energy
cells (Chambers et al., 2017) and neurons of the nucleus of the homeostasis by acting via the CNS, particularly the hypothala-
solitary tract (NTS), which mainly project to the hypothalamus mus (Adams et al., 2018; Beiroa et al., 2014; Secher et al.,
(Holt et al., 2019). However, the rapid degradation of native 2014; Sisley et al., 2014; Timper et al., 2020) and the brainstem
GLP1 by dipeptidylpeptidase 4 (DPP-4) in vivo limits its thera- (Fortin et al., 2020). Initially, liraglutide was thought to reach
peutic potential. This has led to the development of GLP1R ag- target cells in the mediobasal hypothalamus that regulate meta-
onists with a longer half-life that promote a wide range of benefits bolism by crossing the blood-brain barrier (BBB) either at the
including reduced food intake, increased weight loss, and level of microvessels or at circumventricular organs (CVOs)
improved glycemic control, and some of these agonists have such as the median eminence (ME) or the area postrema (AP),
been approved by American and European health agencies for which lie adjacent to the arcuate nucleus of the hypothalamus
the treatment of obesity and type 2 diabetes (T2D) (Drucker (ARH) (Banks, 2019; Mullier et al., 2010; Prevot et al., 2018)
et al., 2017). The GLP1R agonist liraglutide has an amino acid and the NTS (Fortin et al., 2020), respectively. However, its exact
sequence that is 97% identical to that of human GLP1 and route of transport into these areas remains unknown. A recent
1054 Cell Metabolism 34, 1054–1063, July 5, 2022 ª 2022 Elsevier Inc.
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Figure 1. Blood-borne liraglutide does not cross the brain-blood barrier (BBB) at endothelial cells but is transcytosed by tanycytes and has
direct access to putative neurons lying outside the BBB in the ME
(A) Representative photomicrographs of the tuberal region of the hypothalamus showing tanycytic processes (arrows) and cell bodies (arrowheads) labeled by
vimentin (green) and fluorescent liraglutide564 (white), 15 and 60 s after intravenous injection (90 nmol/kg). Note that some neuron-like cell bodies also appear to
internalize liraglutide564 (white arrowheads) and that liraglutide564 does not extravasate from BBB vessels (purple arrows). Scale bar, 200 mm.
(B) Representative images of immunohistochemical staining for the tight junction markers Claudin-5 (green) and ZO-1 in primary tanycytes. Scale bar, 30 mm.
(C) Accumulation of 125I liraglutide with or without unlabeled liraglutide, in endothelial cells of the traditional BBB model (white bars) and in tanycytes (black bars).
Data were analyzed using one-way ANOVA (F(6, 19) = 8.85; p = 0.0001) followed by Tukey’s multiple comparison test (125I-liraglutide BBB versus tanycytic model,
p < 0.0001; 125I-liraglutide BBB without liraglutide versus with liraglutide; p < 0.0001) (n = 3, 3, and 8; 8 wells from two independent experiments).
(D) Competition curve of 125I-liraglutide and increasing concentrations (in nM) of unlabeled liraglutide (n = 6, 3, 3, 3, 4, and 4; 3 wells from two independent
experiments).
(E) Time-dependent accumulation (blue line) and release (red line) of 125I-liraglutide (cellular accumulation n = 4, 3, 3, 3, 6, 3, and 3 wells from two independent
experiments; total release n = 3).
(F) Representative photomicrographs showing pCREB in tanycytes and neuron-like cells in the vmARH of mice after treatment with saline (left panel) versus
liraglutide (right panel). Scale bar, 150 mm (insets, 50 mm).
(G and H) Quantification of pCREB-positive tanycytes (G) and neuron-like cells (H) in the ME, vmARH, dmARH, and ARHtot (n = 4 animals per group; three to four
sections per animal).
(legend continued on next page)
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study in which the AP was surgically resected suggests that this expressed GLP1R (Figure S1FIV), as in vivo (Figure S1G). We
CVO is not required for the brain-mediated effects of liraglutide compared the uptake of 125I-liraglutide (0.7 nM) in the presence
on food intake and body weight (Fortin et al., 2020). On the other or absence of unlabeled liraglutide (10 mM) in the two culture
hand, in the ME, highly specialized ependymoglial cells called ta- models and found that liraglutide accumulation was approxi-
nycytes transport blood-borne metabolic signals such as leptin mately 4 times higher in tanycytes than in endothelial cells (Fig-
and ghrelin into the cerebrospinal fluid (CSF), thus acting as ure 1C). Competition experiments revealed an S-shaped binding
gatekeepers for these molecules (Balland et al., 2014; Collden curve with an apparent KM of 300 nM (Figure 1D), suggesting
et al., 2015; Duquenne et al., 2021; Garcia-Caceres et al., uptake by a single receptor across tanycytes. Subsequently,
2019; Prevot et al., 2018). Due to the privileged position of ME ta- pulse-chase experiments in vitro revealed the rapid uptake of
125
nycytes, which are in direct contact with blood-borne nutrients I-liraglutide by tanycytes and the saturation of the process
and hormones through the fenestrated endothelial walls of ME within 15 min. Upon removal of the radiolabeled substrate, its
capillaries (Langlet et al., 2013b), we hypothesized that these intracellular concentration immediately declined, indicating the
cells could also mediate the entry of liraglutide into the medio- exocytosis of the drug from tanycytes (Figure 1E). Since previous
basal hypothalamus, enabling it to regulate energy homeostasis. in vitro studies have shown that liraglutide activates CREB phos-
phorylation (pCREB) (Athauda and Foltynie, 2016; Que et al.,
RESULTS 2019), we used pCREB as a readout for liraglutide-induced cell
activation. Immunohistochemical labeling revealed that the intra-
Blood-borne liraglutide is transported by tanycytes into venous administration of liraglutide markedly induced pCREB at
the median eminence 1 min in tanycytes and neuron-like cells of the ME and vmARH,
In order to ‘‘catch’’ liraglutide in the process of entering the brain compared to saline-treated animals (Figures 1F–1H). pCREB
and thereby determine its route of access, we fluorescently was also induced in rat primary tanycytes treated with liraglutide,
labeled liraglutide and administered it intravenously through an effect that was blunted by co-treatment with the GLP1R
the jugular vein. We observed that 60 s after its injection, liraglu- antagonist, exendin 9-39 (Figure S2A). Altogether, these obser-
tide did not cross the microvascular endothelial cells of the BBB vations suggest that liraglutide obtains access to hypothalamic
(Figure 1A, purple arrows), but extravasated through the fenes- neurons via GLP1R-expressing tanycytes rather than by
trated vessels of the ME. Its presence in tanycytes and crossing BBB vessels, but may also bind directly to vmARH neu-
neuronal-like cell bodies of the ventromedial ARH (vmARH) at rons residing outside the BBB (Djogo et al., 2016; Schaeffer
this short time point suggests uptake by a direct action on cells et al., 2013; Yulyaningsih et al., 2017).
lying outside the BBB (Figure 1A). Furthermore, in an in vitro
model of the BBB (Figures S1A–S1C) (Helms and Brodin, Transgenic expression of botulinum toxin in tanycytes
2014), neither GLP1 nor liraglutide crossed the BBB at endothe- impedes liraglutide-mediated neuronal activation in the
lial cells, either by diffusion or by GLP1R-mediated uptake (Fig- hypothalamus
ure S1D), possibly because this receptor is not expressed by To determine whether transcytosis across tanycytes mediates
endothelial cells (Figure S1EV). To exclude the possibility that the liraglutide-induced activation of hypothalamic neurons, we
the fluorescent moiety modified the physiological route of liraglu- used a conditional mouse model expressing the botulinum toxin
tide entry into the CNS, we tested the effects of the radioactively serotype B light chain (BoNT/B), permitting the inducible cell-
labeled compounds 125I-GLP1 and 125I-liraglutide. In the BBB specific inhibition of SNARE-mediated exocytosis (Slezak
model, both had lower permeability coefficients (Papp) compared et al., 2012). BoNT/B expression in tanycytes was achieved by
to that of sodium fluorescein, but similar to that of FITC-dextran injecting the recombinant protein TAT-Cre into the third ventricle
(FD-4), two markers of paracellular diffusion. Transcytosed of BoNTB-EGFPloxP-STOP-loxP mice, as described previously
levels of the 125I-GLP1R-agonists (GLP1 or liraglutide) were not (Langlet et al., 2013a). Cre- and vehicle-injected animals are
modified by the presence of an excess of unlabeled liraglutide hereafter referred to as iBot and control mice, respectively (Fig-
or of the GLP1R antagonist, exendin 9-39, further indicating ure 2A). The selective expression of the transgene in tanycytes of
that blood-borne liraglutide does not cross BBB endothelial cells iBot mice was confirmed by the co-expression of GFP and
to reach the brain (Figure S1D). BoNT/B in fluorescence-activated cell sorting (FACS)-sorted
We next investigated the role of tanycytes in liraglutide trans- cells from ME/ARH explants (Figure S2B). Furthermore, these
port using primary cultures in a transwell system (Figures S1FI). cells expressed mRNA for Darpp-32 (a tanycytic marker) and
Similar to the endothelial BBB model (Figure S1B), tanycytes, Glp1r, but, in contrast to GFP-negative cells, did not express
which replace the BBB at CVOs (Langlet et al., 2013b; Mullier Npy (a neuronal marker) or Meca32 (an endothelial cell marker)
et al., 2010), expressed organized tight-junction complexes (Figure S2C). Glp1r expression was comparable in GFP-positive
in vitro, as they do in vivo (Figure 1B). However, in contrast to and GFP-negative cells, i.e., in tanycytes and in other GLP1-sen-
endothelial cells (Figure S1E), primary cultured tanycytes also sitive hypothalamic cells, including neurons (Figure S2D). We
(G) Data were analyzed using an unpaired t test (ME saline versus liraglutide t(6) = 2.40, p = 0.0265; vmARH saline versus liraglutide t(6) = 1.96, p = 0.0491; ARHtot
[vmARH + dmARH] saline versus liraglutide t(6) = 2.15, p = 0.0376).
(H) Data were analyzed using an unpaired one-tailed t test (vmARH saline versus liraglutide t(5) = 4.66, p = 0.0028).
ME, median eminence; ARH, arcuate nucleus of the hypothalamus; vm, ventromedial; dm, dorsomedial; tot, total; VMH, ventromedial nucleus of the hypo-
thalamus; DMH, dorsomedial nucleus; 3V, third ventricle.
Data are represented as means ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001.
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Figure 2. Liraglutide-induced c-Fos activation is abolished in the hypothalamus of iBot mice selectively expressing botulinum toxin in
tanycytes
(A) Protocol for Cre-dependent induction of BoNT/B in tanycytes followed by the injection of recombinant TAT-Cre or vehicle into the third ventricle of BoNTB-
EGFPloxP-STOP-loxP mice.
(B–E) Representative photomicrographs of c-Fos immunohistochemistry in the hypothalamus of control (B and C) and iBot (D and E) mice 1 h after intraperitoneal
injection of saline (left panels) or liraglutide (90 nmol/kg, right panels). AHN, anterior hypothalamic nucleus; ARH, arcuate nucleus of the hypothalamus; DMH,
dorsomedial hypothalamic nucleus; LHA, lateral hypothalamic nucleus; PVH, paraventricular nucleus of the hypothalamus; TUB, tuberal nucleus; ZI: zona incerta.
Scale bar, 200 mm.
(F) Quantification of the number of c-Fos-positive cells in mouse hypothalamic sections 1 h after intraperitoneal injection of either saline (black, gray) or liraglutide
(0.1 mg/kg) (green, blue) in control (black and green) and iBot mice (gray and blue) (n = 3, 4, and 3; 4 animals per group; six to seven sections per animal). PVH:
two-way ANOVA, genotype, F(1, 10) = 6.96, p = 0.024; treatment, F(1, 10) = 0.586, p = 0.024; interaction, F(1, 10) = 6.502, p = 0.0289; Fisher’s LSD post hoc test,
control saline versus control liraglutide, p = 0.0410 and control liraglutide versus iBot liraglutide, p = 0.0027; LHA: two-way ANOVA, genotype, F(1, 10) = 15.54, p =
0.0028; treatment, F(1, 10) = 5.19, p = 0.045; interaction, F(1, 10) = 4.468, p = 0.0607; Fisher’s LSD post hoc test, control saline versus control liraglutide, p = 0.011
and control liraglutide versus iBot liraglutide, p = 0.0009; DMH: two-way ANOVA, genotype, F(1, 10) = 5.21, p = 0.0456; treatment, F(1, 10) = 0.1, p = 0.756;

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next assessed whether intravenously injected liraglutide could selectively blunted in tanycytes of Glp1rTanycyteKD mice (Fig-
stimulate c-Fos expression in the hypothalamus of iBot mice ure 4B). Furthermore, FACS experiments demonstrated that
compared to control littermates. c-Fos expression occurs within the virus-mediated expression of Glp1r shRNA markedly and
minutes after cell activation (Manna and Stocco, 2007), allowing selectively blunted the expression of Glp1r transcripts in GFP-
us to identify first-order GLP1R-expressing cells. Ten minutes af- positive cells (Figure 4C), which expressed mRNA for the
ter the intravenous injection of liraglutide in control anesthetized tanycytic marker Gpr50, but not proopiomelanocortin (Pomc),
mice, neurons in the anterior hypothalamic nucleus (AHN), para- indicating that GLP1R-expressing POMC neurons of the ARH
ventricular nucleus (PVH), and lateral hypothalamic area (LHA) were spared (Figure S4C). Like iBot mice, Glp1rTanycyteKD mice
were activated (Figures S3A, S3B, and S3E). AHN and LHA were resistant to the effects of liraglutide on food intake (Fig-
have not previously been identified as targets of liraglutide treat- ure 4D), body weight reduction (Figure 4E), and fatty acid
ment (Adams et al., 2018; Salinas et al., 2018), although GLP1 oxidation (Figures 4F and 4G). To investigate whether the tany-
binding sites have been documented in these nuclei (Goke cyte-specific blunting of GLP1R expression in Glp1rTanycyteKD
et al., 1995). Notably, liraglutide failed to significantly induce c- mice was linked to an alteration of the GLP1R-dependent trans-
Fos expression in hypothalamic neurons of iBot mice port of blood-borne liraglutide into the hypothalamus, microdial-
(Figures S3C–S3E). One hour after intraperitoneal injection of lir- ysis probes were implanted into the dorsomedial hypothalamus
aglutide, neurons in the PVH and the LHA, but not in the AHN, of AAV1/2-transduced mice (Figure 4H). Thirty minutes after an
were seen to remain activated in control unanesthetized mice intraperitoneal bolus of liraglutide, an ELISA for GLP1 (which
(Figures 2B, 2C, and 2F), whereas c-Fos expression was unaf- also recognizes related peptides such as liraglutide) (Figure S4D)
fected by liraglutide treatment in iBot littermates (Figures 2D– revealed a significant peak in the concentration of these pep-
2F). While liraglutide did not significantly upregulate c-Fos tides in intrahypothalamic dialysates from control mice, but not
expression in the dorsomedial nucleus of the hypothalamus from Glp1rTanycyteKD mice (Figure 4I).
(DMH) or the zona incerta (ZI) in control mice, its expression in Altogether, these results demonstrate that tanycytic GLP1R
these two regions was lower in liraglutide-treated iBot mice binding is required for the transport of blood-borne liraglutide
when compared to liraglutide-treated control littermates into the hypothalamus, where it exerts its effects on food intake
(Figures 2B–2F). Altogether, these results demonstrate that the and lipid metabolism.
blockade of vesicular trafficking in tanycytes blunts acute liraglu-
tide-mediated neuronal activation in the tuberal region of the DISCUSSION
hypothalamus and suggest that circulating liraglutide enters
the hypothalamus via transcytosis through tanycytes. Several studies to date have shown that the effects of liraglutide
on weight loss are predominantly mediated by its actions in the
GLP1R-dependent tanycytic transport is required for brain (Adams et al., 2018; Beiroa et al., 2014; Fortin et al.,
the anti-obesity effects of liraglutide 2020; Secher et al., 2014; Sisley et al., 2014). Although the exact
We next explored the anti-obesity actions of liraglutide in control route of entry of liraglutide into the brain has not been investi-
and iBot littermates after a 3-day treatment (Figure 3A). We first gated, previous studies have reported the direct entry of
found in controls that liraglutide markedly reduced energy blood-borne GLP1 and its analog, exendin-4, into the brain
expenditure (Figure S4A) and food intake (Figure 3B), while pro- across the BBB (Kastin and Akerstrom, 2003; Kastin et al.,
moting body weight loss and fat mass reduction (Figures 3C and 2002). However, our present results indicate that blood-borne lir-
3D) in association with increased fatty acid oxidation (Figure 3E). aglutide does not cross the BBB through endothelial cells, which
However, in iBot mice, these effects were abrogated do not appear to express GLP1R, but by transcytosis through
(Figures 3B–3D and 3F), suggesting that the central metabolic hypothalamic tanycytes, which, in conjunction with the fenes-
actions of liraglutide require tanycyte-mediated access to the trated vessels of the ME, are also components of the BBB (Pre-
hypothalamic neural substrate (Adams et al., 2018; Cavalcanti- vot et al., 2021). This route of entry is in perfect accord with the
de-Albuquerque et al., 2019; He et al., 2019; Secher et al., key role played by the tanycytic shuttle in transporting other pe-
2014). To determine whether GLP1R expression in tanycytes ripheral metabolic signals, such as leptin, ghrelin, and insulin,
(Figure S1G) plays a role in mediating the effects of liraglutide into the mediobasal hypothalamus (Balland et al., 2014; Collden
in the hypothalamus, tanycytic GLPIR expression was specif- et al., 2015; Duquenne et al., 2021; Porniece Kumar et al., 2021).
ically knocked down by the AAV1/2-mediated expression of a The actions of liraglutide in the hypothalamus are extremely
Glp1r-silencing shRNA (Glp1rTanycyteKD mice) and green fluores- rapid—it is internalized by tanycytes in a matter of seconds
cent protein (GFP) (Figure S4B). RNAscope in situ hybridization following its administration and activates responsive neurons in
studies revealed that GLP1R was abundantly expressed in cells deeper nuclei that are shielded by the BBB in a matter of mi-
of the ARH, including in vimentin-immunoreactive tanycytic cell nutes. Interestingly, blocking the tanycytic liraglutide shuttle
bodies lining the wall of the third ventricle and their processes, blunts the activation of deeper neurons as well as changes in
in mice transduced with a control virus (Figure 4A), but was food intake, fatty acid oxidation, and weight loss, indicating the
interaction, F(1, 10) = 3.827, p = 0.078; Fisher’s LSD post hoc test, control liraglutide versus iBot liraglutide, p = 0.0089; ZI: two-way ANOVA, genotype, F(1, 10) =
2.25, p = 0.164; treatment, F(1, 10) = 0.03, p = 0.871; interaction, F(1, 10) = 6.15, p = 0.032; Fisher’s LSD post hoc test, control liraglutide versus iBot liraglutide,
p = 0.0124.
Data are expressed as means ± SEM. *p < 0.05; **p < 0.01, control saline versus control liraglutide; #p < 0.05; ##p < 0.01, control liraglutide versus iBot liraglutide.

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Figure 3. The anti-obesity effects of liraglutide are abolished by using botulinum toxin expression to block vesicular transport in tanycytes
(A) Experimental setup to measure the metabolic effects of liraglutide.
(B) Cumulative food intake in the different light/dark phases 3 days after liraglutide treatment, compared to baseline (night phase: two-way ANOVA, genotype,
F(1, 28) = 1.64, p = 0.210; treatment, F(1, 28) = 42.36, p < 0.0001; interaction, F(1, 28) = 13.69, p = 0.0009; Tukey’s post hoc test, control saline versus control
liraglutide, p < 0.0001 and iBot saline versus iBot liraglutide, p = 0.240; sum: two-way ANOVA, genotype, F(1, 28) = 2.63, p = 0.116; treatment, F(1, 28) = 16.59,
p = 0.0003; interaction, F(1, 28) = 10.69, p = 0.0029; Tukey’s post hoc test, control saline versus control liraglutide, p < 0.0001 and iBot saline versus iBot
liraglutide, p = 0.945) (n = 8, 9, 8, and 7 mice).
(C) Body weight change after the experiment shown in (A) (two-way ANOVA, genotype, F(1, 31) = 8.98, p = 0.53; treatment, F(1, 31) = 32.85, p < 0.0001; interaction,
F(1, 31) = 1.01, p = 0.324; Tukey’s post hoc test, control saline versus control liraglutide, p = 0.0001 and iBot saline versus iBot liraglutide, p = 0.0154; control
liraglutide versus iBot liraglutide, p = 0.042) (n = 10, 9, and 8; 8 mice).
(D) Fat mass change after the experiment shown in (A). Data were analyzed using an unpaired one-tailed t test (fat mass, t(18) = 2.53, p = 0.0104; body weight
change, p = 0.0495) (n = 10; 10 mice).
(E and F) Fatty acid oxidation and area under the curve (AUC) in control (E) and iBot (F) animals 3 days after liraglutide injection, compared to saline (n = 8 mice per
group). Two-way ANOVA with Tukey’s post hoc test (control saline versus control liraglutide, 20.00, p = 0.0191; 21.00, p = 0.0311; 22.00, p = 0.0061; 2.00, p =
0.0408; 4.00, p = 0.0066) (n = 9; 8 mice). Red dotted lines indicate liraglutide or vehicle administration. AUC, paired two-tailed t test, t(5) = 3.62, p = 0.0152, n = 6
mice in (E) and t(5) = 2.39, p = 0.0624, n = 6 mice in (F). iBot saline versus iBot liraglutide, #p < 0.05; yp < 0.05 control liraglutide versus iBot liraglutide, *p < 0.05,
**p < 0.01, ***p < 0.001, ****p < 0.0001.
critical importance of the tanycyte-mediated access of liraglu- meostasis and pinpoints the role of tanycytes, which express
tide to hypothalamic nuclei protected by the BBB for its meta- GLP1R and actively transport blood-borne liraglutide into the
bolic effects. In fact, even though liraglutide directly activates brain, in this process. Our results further cement the position of
pCREB in neurons bordering the ME by extravasation from these unique cells, which act as a bridge between the peripheral
fenestrated vessels, in order to exert its effects on neurons pro- circulation and the brain, as master regulators of the body’s en-
tected by the BBB, such as those of the dorsomedial ARH, its ergy/metabolic status.
transport across the BBB by tanycytes is essential. This dual
mode of access is also similar to that observed with leptin (Duqu- Limitations of study
enne et al., 2021). Moreover, by knocking down GLP1R specif- In order to identify the route of entry of liraglutide by catching it in
ically in tanycytes, we demonstrate that liraglutide transcytosis flagrante delicto, in the process of crossing the BBB, and deter-
into the brain and its anti-obesity effects require tanycytic mine the consequences of its action through its immediate target
GLP1R binding and the activation of its downstream signaling neurons in the hypothalamus, we limited our studies to a 3-day
cascade, again evoking the receptor-mediated transcytotic treatment. However, while this was sufficient to identify the tany-
mechanism involved in leptin transport (Duquenne et al., 2021). cytic shuttle as essential for liraglutide transport into the brain,
It remains to be seen how tanycytic barrier properties, which and while a previous study has shown that the effects of liraglu-
are modulated by energy status (Langlet et al., 2013a), and their tide on food intake are attenuated after the first 24 h (Fortin et al.,
transport of GLP1R agonists may themselves be altered by 2020; Killion et al., 2018), it is entirely possible that some of these
obesity (or T2D), thus affecting the entry and efficacy of the effects are cumulative (Secher et al., 2014; Sisley et al., 2014) or
very drugs needed to treat it. that the long-term effects of GLP1R agonists on food intake
To conclude, our study reveals that liraglutide acts directly on through other brain areas, such as the AP or the NTS (Brierley
its target neurons in the hypothalamus to improve energy ho- et al., 2021; Fortin et al., 2020), contribute to their efficacy.

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Figure 4. Knocking down GLP1R in tanycytes abolishes both the shuttling of blood-borne liraglutide into the hypothalamus and its anti-
obesity effects
(A and B) Representative photomicrographs of the tuberal region of the hypothalamus showing vimentin-immunoreactive tanycytic processes and cell bodies
(red) and GLP1R mRNA expression (white dots) after transduction of tanycytes with a control AAV1/2 (A) or an AAV1/2-expressing GLP1R shRNA (B). Empty
arrowheads indicate tanycytic cell bodies expressing GLP1R in the vmARH and white arrows point to tanycytic processes where vimentin and Glp1-r transcripts
are colocalized. Scale bar, 200 mm.
(C) Schematic diagram: sorting of GFP-positive putative tanycytes following AAV1/2 control or AAV1/2 shRNA-GLP1R infusion into the lateral ventricle. Bar
graph: expression of GLP1R mRNA in GFP-positive and -negative FACS-sorted cells. Unpaired one-tailed t test (positive cells; control versus GLP1RtanycteKD,
t(7) = 2.08, p = 0.0377, n = 4; 5 mice).
(D) Cumulative food intake during the different light/dark phases 3 days after liraglutide treatment, compared to baseline (night: two-way ANOVA, genotype,
F(1, 27) = 7.88, p = 0.009; treatment, F(1, 27) = 3.59, p = 0.069; interaction, F(1, 27) = 4.72, p = 0.039; Tukey’s post hoc test, control saline versus control lir-
aglutide, p = 0.033 and Glp1-rTanycyteKD saline versus Glp1-rTanycyteKD liraglutide, p = 0.99; control liraglutide versus Glp1-rTanycyteKD liraglutide, p = 0.042; sum:
two-way ANOVA, genotype, F(1, 27) = 7.64, p = 0.010; treatment, F(1, 27) = 1.12, p = 0.299; interaction, F(1, 27) = 5.48, p = 0.027; Tukey’s post hoc test, control
saline versus control liraglutide, p = 0.09 and Glp1-rTanycyteKD saline versus Glp1-rTanycyteKD liraglutide, p = 0.809; control liraglutide versus Glp1-rTanycyteKD
liraglutide, p = 0.007; n = 8, 8, and 8; 7 mice).
(E) Body weight change after the experiment (two-way ANOVA, genotype, F(1, 26) = 3.07, p = 0.091; treatment, F(1, 26) = 11.48, p = 0.0022; interaction, F(1, 26) = 2.47,
p = 0.128; Tukey’s post hoc test, control saline versus control liraglutide, p = 0.0017 and Glp1-rTanycyteKD saline versus Glp1-rTanycyteKD liraglutide, p = 0.210;
control liraglutide versus Glp1-rTanycyteKD liraglutide, p = 0.031, n = 8, 7, and 8; 7 mice).

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Further evaluation of the effects of liraglutide or other GLP1R ag- AUTHOR CONTRIBUTIONS
onists at longer time points is necessary to completely under- M.I. and V.P. conceived the study. M.I., C.S., H.C.C.H., M.D., D.F., and E.D.
stand the mode of action of these drugs on the brain. carried out the experiments. H.C.C.H. and B.B. set up the classical BBB model
and performed in vitro barrier experiments. M.I., R.G.P.D., D.H.M.C., and S.L.
STAR+METHODS conducted metabolic phenotyping. D.F. and M.I. performed microdialysis ex-
periments. F.W.P. generated the iBot animal model. M.I., C.S., B.S., F.P., S.L.,
C.B., and V.P. designed and planned the experiments. M.I., C.S., C.B., and
Detailed methods are provided in the online version of this paper
V.P. wrote the paper. S.R. edited the manuscript. All authors contributed to
and include the following: the preparation of the manuscript.
d KEY RESOURCES TABLE

d RESOURCE AVAILABILITY DECLARATION OF INTERESTS
B Lead contact
The authors declare no competing interests.
B Materials availability
B Data and code availability Received: August 31, 2020
d EXPERIMENTAL MODEL AND SUBJECT DETAILS Revised: August 8, 2021
B Animals Accepted: June 1, 2022
B Primary cultures Published: June 17, 2022
d METHOD DETAILS
B Stereotaxic TAT-Cre delivery REFERENCES
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SUPPLEMENTAL INFORMATION
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ACKNOWLEDGMENTS 258–273. https://doi.org/10.1038/s42255-021-00344-4.
Bruss, M.D., Khambatta, C.F., Ruby, M.A., Aggarwal, I., and Hellerstein, M.K.
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grant no. 810331 to V.P., H2020-MSCA grant no. 748134 to M.I., the Agence oxidation rates. Am. J. Physiol. Endocrinol. Metab. 24, E108–E116. https://
National de la Recherche (ANR, France) grant ANR-15-CE14-0025 to V.P., Eu- doi.org/10.1096/fasebj.24.1_supplement.336.8.
ropean Genomic Institute for Diabetes (EGID, ANR-10-LABX-0046 and I-SITE
Cavalcanti-de-Albuquerque, J.P., Bober, J., Zimmer, M.R., and Dietrich, M.O.
ULNE ANR-16-IDEX-0004 to V.P., F.P., and B.S.), the Conseil Regional Nord-
(2019). Regulation of substrate utilization and adiposity by Agrp neurons. Nat.
Pas de Calais (to C.B.), and funding from Novo Nordisk A/S (S.L. and V.P.).
Commun. 10, 311. https://doi.org/10.1038/s41467-018-08239-x.
H.C.C.H. was supported by a fellowship from Novo Nordisk A/S. B.S. is a
recipient of an ERC Advanced Grant (694717). We thank Marc R. Montminy Chambers, A.P., Sorrell, J.E., Haller, A., Roelofs, K., Hutch, C.R., Kim, K.S.,
(Salk Institute, La Jolla, CA, USA) for his generous gift of the phopho-CREB Gutierrez-Aguilar, R., Li, B., Drucker, D.J., D’Alessio, D.A., et al. (2017). The
antibody. We thank Emilie Caron (metabolic cages), Nathalie Jouy and Aman- role of pancreatic preproglucagon in glucose homeostasis in mice. Cell
dine Legrand (cell sorting), Antonino Bongiovani (imaging facility) from the Metab. 25, 927–934.e3. https://doi.org/10.1016/j.cmet.2017.02.008.
BioImaging Center of Lille (BiCeL), and Julien Devassine (animal core facility) Collden, G., Balland, E., Parkash, J., Caron, E., Langlet, F., Prevot, V., and
of the PLBS UAR2014-US41 for their expert technical support. Bouret, S.G. (2015). Neonatal overnutrition causes early alterations in the
(F and G) AUC of 24 h fatty acid oxidation in control (F) and Glp1rtanycyteKD (G) animals 3 days after liraglutide injection, compared to saline (n = 8 mice per group).
Paired two-tailed t test t(6) = 3.62, p = 0.011 in (F) (n = 7 mice) and t(6) = 0.02, p = 0.972 in (G) (n = 8 mice).
(H) Schematic diagram illustrating the implantation of the microdialysis probe in the mediobasal hypothalamus.
(I) GLP1 concentrations in the ARH interstitial liquid collected by microdialysis every 15 min following intraperitoneal liraglutide (t0 min) injection (0.1 mg/kg) in
control (n = 6) and GLP1RtanKO mice (n = 5). Two-way ANOVA followed by a Bonferroni post hoc test, p = 0.0088. AUC, one-tailed t test, t(9) = 2.10, p = 0.0323. Data
are expressed as means ± SEM. *p < 0.05; **p < 0.01, control saline versus control liraglutide and control versus Glp1rtanycyteKD; yp < 0.05 control liraglutide versus
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STAR+METHODS
KEY RESOURCES TABLE
REAGENT or RESOURCE SOURCE IDENTIFIER

Antibodies
Rabbit polyclonal anti-mouse/human Claudin-5 Thermo Fisher Cat#: 34–1600; RRID: AB_2533157
Rabbit monoclonal anti-mouse/human/rat Cell Signaling Cat#: 9198; RRID: AB_2561044
Phospho-CREB (Ser1322)
Rabbit polyclonal anti-mouse/human/rat/cat/ DakoCytomation Cat#: Z0334; RRID: AB_10013382
dog/cow/sheep GFAP
Mouse polyclonal anti-mouse/human/rabbit/rat GLP1-R Novo Nordisk Cat#: MAB 7f38; RRID: AB_2618101
Chicken polyclonal anti-mouse/human/rat/ Merck Millipore Cat#: AB5733; RRID: AB_11212377
chicken/cat Vimentin
Rabbit polyclonal anti-human von Willebrand’s Factor Sigma-Aldrich Cat#: F3520; RRID: AB_259543
Rabbit polyclonal anti-mouse PECAM-1 Santa Cruz Biotechnology Cat#: sc1506; RRID: AB_2161037
Mouse monoclonal anti-human/rat/dog/zebrafish ZO1 Thermo Fisher Cat#: 33–9111; RRID: AB_87182
Rabbit polyclonal anti-mouse phospho-CREB Hagiwara et al. (1993); N/A
Sanchez et al. (2010)
Rabbit polyclonal anti-mouse/human/rat c-FOS Synaptic Systems Cat#: 226008; RRID: AB_2231974
Rabbit polyclonal anti-mouse GFP Thermo Fisher Cat#: A-11122; RRID: AB_221569
Biotin-SP polyclonal goat anti-rabbit IgG Jackson ImmunoResearch Cat#: 111-065-144; RRID: AB_2337965
Goat polyclonal anti-rabbit IgG, Alexa Fluor 488 Invitrogen Cat#: A11008; RRID: AB_143165
Goat polyclonal anti-rabbit IgG, Alexa Fluor 568 Invitrogen Cat#: A11011; RRID: AB_143157
Goat polyclonal anti-mouse IgG, Alexa Fluor 488 Invitrogen Cat#: A11002; RRID: AB_1500639
Goat polyclonal anti-mouse IgG, Alexa Fluor 568 Invitrogen Cat#: A11031; RRID: AB_144696
Goat polyclonal anti-chicken IgG, Alexa Fluor 568 Invitrogen Cat#: A11041; RRID: AB_2534098
Goat polyclonal anti-chicken IgG, Alexa Fluor 488 Invitrogen Cat#: A11039; RRID: AB_142924
Goat polyclonal anti-chicken IgG, Alexa Fluor 647 Invitrogen Cat#: A-21449; RRID: AB_2535
Bacterial and virus strains
AAV1/2-EGFP-U6-mGLP1R-shRNA Vector Biolabs N/A
AAV1/2-EGFP-U6(SapI) Vector Biolabs N/A
Chemicals, peptides, and recombinant proteins
TAT-Cre fusion protein Peitz et al. (2002) N/A
125
I-GLP-1 Novo Nordisk N/A
125
I-liraglutide Novo Nordisk N/A
Fluorescent Liraglutide564 Novo Nordisk N/A
Liraglutide Novo Nordisk N/A
Alexa 488-GLP-1 Novo Nordisk N/A
Exendin 9-39 Novo Nordisk N/A
CMA Perfusion Fluid CNS CMA Microdyalisis Cat#: CMAP000151
RNaseOUT Thermo Fisher Cat#: 10777019
DMEM Sigma-Aldrich Cat#: D6429
DMEM/F12 Sigma-Aldrich Cat#: D6434
DMEM without sodium pyruvate Sigma-Aldrich Cat#: D5796
DMEM without NaHCO3 Sigma-Aldrich Cat#: D5648
Penicillin-streptomycin Gibco Cat#: 15140122
Insulin Sigma-Aldrich Cat#: I-5500
Donor Bovine Serum Gibco Cat#: 16030-074
Mouse endothelial cell culture supplement kit PeloBiotech Cat#: PB-M1168-KIT
(Continued on next page)
e1 Cell Metabolism 34, 1054–1063.e1–e7, July 5, 2022

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Continued
REAGENT or RESOURCE SOURCE IDENTIFIER
Hank’s balanced salt solution Gibco Cat#: 14287
HEPES Gibco Cat#: 11560496
L-glutamine Gibco Cat#: 25030-061
Bovine insulin Sigma-Aldrich Cat#: I6634
Putrescine Sigma-Aldrich Cat#: P5780
BSA Sigma-Aldrich Cat#: A7030
Ovoalbumin Sigma-Aldrich Cat#: A5503
Dexamethasone Merck Millipore Cat#: D4902
Ultima Gold scintillation fluid Perkin-Elmer Cat#: 6013321
MEM Non-Essential Amino Acids Solution Gibco Cat#: 11140050
Triton X-100 Sigma-Aldrich Cat#: T8787
8-(4-CPT)-cyclic adenosine monophosphate Merck Millipore Cat#: C0735
Alexa-Fluor 568-streptavidin Invitrogen Cat#: S11226; RRID: AB_2315774
FITC-dextran Sigma-Aldrich Cat#: 46944
Dextran Sigma-Aldrich Cat#: D8821
Sodium fluorescein Merck Millipore Cat#: F6377
Collagen IV Merck Millipore Cat#: CC076
Merck Millipore Cat#:FIBRP-RO
Ro-20-1724 Calbiochem Cat#: 557502
Hoechst 33,258 Invitrogen, Cat#: 10778843; RRID: AB_2651133
DAPI BD Biosciences Cat# 564907; RRID: AB_2869624
Mowiol Merck Millipore Cat#: 475904
Normal goat serum Sigma-Aldrich Cat#: D9663; RRID: AB_2810235
Critical commercial assays
Papain Dissociation System Worthington Papain Cat#: LK003150
TaqMan Pre-Amp Master Mix Kit Applied Biosystems Cat#: 4366128
Super-Script III Reverse Transcriptase Thermo Fisher Cat#: 18080093
Northern Lights Mercodia Total GLP-1 NL-ELISA Mercodia Cat#: 10-1278-01; RRID: AB_2892202
RNAscope Multiplex Fluorescent Kit Advanced Cell Diagnostics Cat#: 320850
Deposited data
Data S1 This study N/A
Experimental models: Organisms/strains
Mus musculus: tdTomatoloxPSTOP-loxP The Jackson Laboratory Stock No.007914; RRID: MSR_JAX:007914
Mus musculus: Tg(CAG-BoNT/B,EGFP)U75-56wp/J The Jackson Laboratory Stock No. 018056; RRID: IMSR_JAX:018056
Mus musculus: C57Bl/6J Charles River RRID: MGI:5657312
Rattus norvegicus: Sprague Dawley Janvier Laboratories RRID: MGI:5651135
Software and algorithms
ImageJ National Institutes of Health RRID: SCR_003070
Photoshop CC Adobe Systems RRID: SCR_014199
Adobe Illustrator CC Adobe Systems RRID: SCR_010279
GraphPad PRISM 8.0 GraphPad Software RRID: SCR_002798
RESOURCE AVAILABILITY
Lead contact
Additional information and requests for reagents and resources should be directed to and will be fulfilled by the lead contact,
Dr. Vincent Prevot (Vincent.prevot@inserm.fr).
Materials availability
This study did not generate new unique reagents.
Cell Metabolism 34, 1054–1063.e1–e7, July 5, 2022 e2

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Data and code availability

d Data S1. Unprocessed data underlying the display items in the manuscript, related to Figures 1, 2, 3, 4, and S1–S4.
d This paper does not report original code
d Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon reason-
able request.
EXPERIMENTAL MODEL AND SUBJECT DETAILS
Animals
All C57Bl/6J mice and Sprague-Dawley rats were housed under specific pathogen-free conditions in a temperature-controlled
room (21–22 C) with a 12h light/dark cycle and ad libitum access to chow diet (Scientific Diets, SAFE A03) and water. Health
status checks of the animal house were performed 4 times per year by the animal facility staff to assure the pathogen-free status.
tdTomatoloxPSTOP-loxP, C57BL/6J background, (Stock No.007914; RRID: IMSR_JAX:007914) were purchased from the Jackson
Laboratories (Bar Harbor, ME, USA) and Tg(CAG-BoNT/B,EGFP)U75-56wp/J, C57BL/6J background, mice have been engineered
by Dr. Franck Pfrieger (University of Strasbourg, France; JAX Stock No. 018056; (RRID: IMSR_JAX:018056) as previously described
(Slezak et al., 2012). Transgenic model mice were bred and genotyped in house to generate experimental animals. Control male
C57Bl/6J (RRID: MGI:5657312) mice were purchased from Charles River. Male Sprague Dawley (RRID: MGI:5651135) rats were pur-
chased from Janvier laboratories (France) or Taconic (Denmark). All experiments were performed in 8–10 weeks-old male mice and in
P2-P10 rat pups of both sexes and 8 weeks-old male rats. All animals were habituated to handling and experimental conditions prior
to experimentation and littermate controls were used.
Animal studies were performed with the approval of the Institutional Ethics Committees for the Care and Use of Experimental An-
imals of the University of Lille and the French Ministry of National Education, Higher Education and Research (APAFIS#2617–
2015110517317420 v5) and under the guidelines defined by the European Union Council Directive of September 22, 2010 (2010/
63/EU).
Primary cultures
Astrocytes
Rat primary astrocytes were isolated from the cerebral cortices of 2–4 days old Sprague-Dawley rat pups as previously described
(Hertz et al., 1989). In brief, the rat pups were decapitated and the pooled cortices were pushed through an 80 mm nylon net filter
(Millipore) into Dulbecco’s modified Eagle’s medium (DMEM) with 20% fetal bovine serum. The cell suspension was cultured in
T75 culture flasks (2 flasks per brain) until confluent. Serum content was gradually reduced to 15 and 10% through a period of three
weeks. During the final week, the cell culture medium was harvested as astrocyte-conditioned medium (ACM). Cells were passaged,
frozen at 80 C and subsequently stored in liquid nitrogen.
Brain capillaries
Primary brain capillaries were isolated from the brain cortices of calves below 12 months (Mogens Nielsen Kreaturslagteri A/S), and
from rat and mice cerebral cortices using the same techniques of recovering the microvessels form gray matter suspension, but
adapted to the size of the different species as previously described (Helms and Brodin, 2014). Capillaries were frozen in aliquots
(10 per brain) and stored in liquid nitrogen until their use in culture. In brief, rat brain capillaries were isolated from 8 weeks old
male Sprague-Dawley rats. Six cortices were isolated using blunt dissection and the meninges were removed by rolling the cortices
on filter paper. The cortices were put into DMEM (5 mL per cortex) and homogenized using a 7 mL Dounce Tissue Grinder (Wheaton
Science Products). The homogenates were mixed 1:1 with a 32% (w/v) dextran solution (60,000–76,000 kDa, Sigma-Aldrich, D8821),
and centrifuged (1500 g, 15 min). The capillary enriched pellet was washed once in DMEM and filtered through 80 mm nylon net filters
(Millipore). The capillaries were digested for 30 min with papain dissociation system according to the manufacturer’s protocol (Wor-
thington Biochemical). The digested microvessels were seeded directly in culture flasks (one T75 for six rat cortices) pre-coated with
collagen IV and fibronectin (10 mg/mL of both; Millipore CC076 and FIBRP-RO, respectively).
Tanycytes
Primary cultures of tanycytes were generated by isolating the median eminence of 10-day-old Sprague-Dawley rat pups. After me-
dian eminence dissociation, cells were incubated with selective tanycyte medium as previously described (Prevot et al., 2003). In
brief, thirty rat pups were decapitated and the median eminences were microdissected and pooled into DMEM +10% donor bovine
serum. The collected median eminences were pushed through a 20 mm nylon net filter and the resulting cell suspension was seeded
in a T75 flask and cultured for 10 days before initial change of culture medium. Medium was then changed every 2–3 days for an
additional week of culture. Cells were passaged and seeded on permeable supports 17 days after isolation.
METHOD DETAILS
Stereotaxic TAT-Cre delivery

Tanycyte-specific genetic modification was performed with the Cre/LoxP system by stereotaxic infusion of a TAT-Cre fusion
protein as previously described (Peitz et al., 2002). TAT-cre or vehicle was stereotaxically infused into the third ventricle (2 mL;

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at 0.2 mL/min; anteroposterior, 1.7 mm; midline, 0 mm; dorsoventral, 5.6 mm) of isoflurane-anesthetized tdTomatoloxPSTOP-loxP or
BoNTB-EGFPloxP-STOP-loxP mice 2 weeks before commencing the experiments.
GLP1R knockdown in tanycytes

Tanycytic specific knockdown of GLP1R was performed in isoflurane-anesthetized 8-weeks old wild type C57Bl/6J male mice
(Charles Rives), by stereotaxically injection of either AAV1/2-shRNA-GLP1R (AAV1/2-EGFP-U6-mGLP1R-shRNA (ACC GCG TCA
ACT TTC TTA TCT TC ACT CGA GTG AAG ATA AGA AAG TTG ACG C TTTTT); serotype 1:2 chimeric; titer = 5.7 x 1010; Vector Bio-
labs) or AAV1/2-GFP (AAV1/2-EGFP-U6(SapI); serotype 1:2 chimeric; titer = 6.5 x 1010; Vector Biolabs) in the lateral ventricle (2 mL; at
0.2 mL/min; anteroposterior, 0.3 mm; midline, 1 mm; dorsoventral, 2.5 mm), 2 weeks before starting the experiments.
In vivo brain microdialysis

Male control and GLP1RtanKO between 30 and 35g were deeply anesthetized with isoflurane (3% in 1 L/min air flow) in an induction
chamber, placed in a stereotaxic apparatus equipped with a mask to maintain anesthesia during all the experiment (isoflurane 1% in
0.7 L/min air flow). Body core temperature was maintained at 37 C with an electrical blanket controlled by a thermostat. A microdial-
ysis cannula (CMA7, 6 kDa, 2 mm membrane length; CMA microdialysis AB, Sweden) was stereotaxically implanted in the medio-
basal hypothalamic area (antero-posterior: 1.4; midline; 0.3 dorsoventral: 6.2 mm). Microdialysis probe was first perfused
with sterile artificial cerebrospinal fluid (CMA Perfusion Fluid CNS. NaCl 147 mmol/L, KCl 2.7 mmol/L, CaCl2, 1.2 mmol/L and
MgCl2 0.85 mmol/L; CMA, Stockholm, Sweden) at a rate of 2 mL/min using a microinjection pump (CMA 402; CMA). Following a sta-
bilization period of 55 min, one dialysate of 15 min was collected to use as a basal reading. At the end of the basal collection,
liraglutide (0.1 mg/kg) was injected intraperitoneally and five dialysates were collected at 15 min intervals . Brain dialysates were
placed in a fraction collector (CMA/820) during the experiment and immediately stored at 80 C until analysis. At the end of the
experiment, mice were euthanized by decapitation and brains immersion-fixed in 4% paraformaldehyde in 0.1M PBS pH7.4.
80-mm brain section were collected using vibratome and counterstained with DAPI (BD Biosystems) to verify probe implantation;
GFP immunohistochemistry was performed to verify accuracy of the AAV1/2 infection.
Intrajugular administration of liraglutide

Under ketamine-xylazine anesthesia (100 mg/kg, 20 mg/kg respectively), an intravenous catheter (Optiva Jelco IV Catheter
Radiopaque, 24G, REF5063) was implanted in the dissected right branch of the jugular vein of 8–10 weeks year old male mice. Fluo-
rescent (liraglutide564) or non-fluorescent liraglutide (both provided by Novo Nordisk) was injected into the jugular vein (90 nmol/kg,
50 mL), and animals were euthanatized at different time points by either decapitation, and the brain post-fixed in PBS 0.1M4% para-
formaldehyde (PFA, pH 7.4) for 4h to study de distribution of liraglutide564 , or transcardially perfused with 20 mL of saline followed by
either borate 0.1M-4% PFA buffer (pH 9.5) or 0.1M PBS-4% PFA buffer (pH 7.4), for c-Fos and pCREB immunohistochemistry
respectively. Following perfusion, brains were collected and post-fixed in the same fixative for 2 to 4 h at 4 C, cryoprotected with
PB 20% sucrose, and embedded in OCT embedding medium (Tissue-Tek), frozen in isopentane, cooled with dry ice, and stored
at 80 C until coronal cryosectioning. Series of coronal sections through the rostro-caudal axis were collected using cryostat (Leica
CM3050S; Leica Microsystems). For liraglutide564 injected animals, 18 mm-thick brain sections were collected on Superfrost/Plus
glass slides (Fisher Scientific); for animals injected with non-fluorescent liraglutide, 30 mm-thick brain sections were collected free
floating in 0.01M PBS before being subjected to c-Fos and pCREB immunohistochemistry . Slide-adhered brain sections were desic-
cated at room temperature for 4 h and stored at 80 C. Free-floating sections were kept at -20 C in anti-freezer solution until pro-
cessed for double-labeling immunofluorescence.
Indirect calorimetry study

Mice were individually housed and acclimatized to the cages for 48h before experimental measurements in a 12h light/dark cycle
(lights from 07:00 to 19:00 h) and an ambient temperature of 22 ± 1 C. Thereafter all mice were injected at 14h with saline
(200 mL) during three consecutive days and then injected with liraglutide (Sisley et al., 2014) (0.1 mg/kg) for three additional consec-
utive days. Mice were analyzed for total energy expenditure, oxygen consumption and carbon dioxide production, food intake using
calorimetric cages (Labmaster, TSE Systems GmbH) (Tschöp et al., 2012). Fat oxidation was calculated as described before (Bruss
et al., 2010), using energy expenditure, oxygen consumption and carbon dioxide production. Mice were monitored daily for body
weight and body composition at the beginning and the end of the experiment. Body mass composition (lean tissue mass, fat
mass, free water and total water content) was analyzed using a nuclear magnetic resonance (NMR) scanner (Minispec mq series,
Bruker) according to manufacturer’s instructions.
Isolation of tanycytes by FACS

The ME from TAT-Cre injected tdTomatoloxP/+, BoNTB-EGFPloxP/+ and AAV1/2 GFP control or AAV1/2 GFP-shRNA-GLP1R injected
mice were microdissected, and enzymatically dissociated using Papain Dissociation System (Worthington Papain) to obtain single-
cell suspensions as described before (Messina et al., 2016). Fluorescence-Activated Cell Sorting (FACS) experiments were per-
formed using an EPICS ALTA Cell Sorter Cytometer device (Beckman Coulter). The cell sort decision was based on measurements
of tdTomato fluorescence or EGFP fluorescence (Tomato: excitation 488nm, detection: bandpass 675 ± 20nm; EGFP: excitation:
488 nm; 50 mW; detection: EGFP bandpass 530/30 nm, autofluorescence bandpass 695/40 nm) by comparing cell suspensions

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from non-infected brain sites (the cortex) and infected brain sites (median eminence). For each animal, 4000 cells tdTomato positive
or 200 cells EGFP positive and negative cells were sorted directly into 10mL extraction buffer: 0.1% Triton X-100 (Sigma-Aldrich) and
0.4 U/ml RNaseOUT (Thermo Fisher).
Quantitative RT-PCR analyses

For gene expression analyses, mRNAs obtained from FACS-sorted tanycytes, were reverse transcribed using Super-Script III
Reverse Transcriptase (Thermo Fisher) and a linear preamplification step was performed for the sorted cells using the TaqMan
Pre-Amp Master Mix Kit protocol (P/N 4366128, Applied Biosystems). Real-time PCR was carried out on Applied Biosystems
7900HT Fast Real-Time PCR System (RRID: SCR_018060) using exon-boundary-specific TaqMan Gene Expression Assays:
GLP1R (Mm00445292_m1), DARPP32 (Ppp1r1b_Mm00454892_m1), GPR50 (Mm00439147_m1), NPY (NPY-Mm03048253_m1),
POMC (Mm00435874_m1) and MECA32 (Plvap-Mm00453379_m1). Control housekeeping genes: r18S (18S-Hs99999901_s1);
ACTB (Actb-Mm00607939_s1). Gene expression data were analyzed using SDS 2.4.1 and Data Assist 3.0.1 software (Applied
Biosystems).
Hormone measurements
Microdialysis samples were analyzed using Northern Lights Mercodia Total GLP-1 NL-ELISA (Mercodia, Cat# 10-1278-01, RRID:
AB_2892202) according to the manufacturer’s instructions.
Establishment of in vitro BBB and tanycyte-models

Bovine endothelial/rat astrocyte BBB vessel model
Frozen bovine brain capillaries from liquid nitrogen storage were thawed and cultured for 4 days (37 C, 10% CO2) in DMEM-Comp
(D6429 medium, Sigma-Aldrich): ACM (1:1) supplemented with 125 mg/mL heparin in collagen type IV/fibronectin coated T75 flasks.
For the first two days after thawing, the medium was supplemented with 4 mg/mL puromycin to kill pericytes. The endothelial cells
were passaged with a brief trypsinization and seeded on collagen IV/fibronectin coated Transwell polycarbonate permeable supports
(90,000 cells/cm2, Area = 1.12 cm2, pore radius = 0.4 mm, Corning Life Sciences). Astrocytes were seeded on the bottom of the sup-
ports two days prior to endothelial cell seeding (120,000 cells/cm2). The co-cultures were cultured for three days in DMEM-Comp +
125 mg/mL heparin under a humid atmosphere of 5% CO2 and 95% air at 37 C, followed by three days of culture in differentiation
medium consisting of DMEM without NaHCO3- (D5648 medium, Sigma-Aldrich), supplemented with 10% fetal bovine serum
(Gibco), 1% (v/v) non-essential amino acid mixture, 100 U/ml - 100 mg/mL penicillin-streptomycin solution, 312.5 mM 8-(4-CPT)-
cyclic adenosine monophosphate (all Gibco), 0.5 mM dexamethasone (all Merck Millipore), 17.5 mM RO-20-1724 (Calbiochem)
and 50 mM 4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES, all Gibco).
Endothelial rat/mouse BBB vessel model
Endothelial cells from rat and mouse brains were cultured for 4–5 days in a culture flask, in mouse endothelial cell culture medium
using the supplement kit (PB-M1168-KIT, PeloBiotech), which contained 4 mg/mL puromycin for the two first days. Endothelial cells
were passaged similar to the bovine cells and seeded in monocultures on coated permeable supports (90,000 cells/cm2). The endo-
thelial cells were cultured for two days in mouse endothelial cell culture medium (with supplement kit) followed by three days in dif-
ferentiation medium as described (Helms and Brodin, 2014), under a humid atmosphere of 5% CO2 and 95% air at 37 C.
BBB tanycytes model
Tanycytes were passaged and seeded in monocultures on coated permeable supports (90,000 cells/cm2) 17 days after isolation
similar to the endothelial cells. The tanycytes were then cultured for four days in DMEM (D5796 medium, Sigma-Aldrich) comple-
mented with 10% donor bovine serum, followed by three days in serum-free medium consisting of DMEM/F12 (D6434 medium,
Sigma-Aldrich) with 15 mM HEPES, 1% penicillin/streptomycin, 1% L-glutamine (all Gibco), bovine insulin (5 mg/mL) and putrescine
(100 mM) (all Sigma-Aldrich), under a humid atmosphere of 5% CO2 and 95% air at 37 C.
TEER and transport assessments

Transcellular transport studies were performed in the BBB models on day 6 on a permeable support. The transendothelial electrical
resistance (TEER) was measured at room temperature prior to all experiments, using an Endohm-12 cup electrode chamber (World
Precision Instruments) connected to a Millicell-ERS device (Millipore). The culture medium was replaced with pre-heated uptake
buffer (as described above) and the cells were added to a solution containing 125I-GLP-1 (0.5 nM, 1 mCi/mL), 125I-liraglutide
(0.7 nM, 1 mCi/mL), 4 kDa FITC-dextran (10 mg/mL) (Sigma-Aldrich) or sodium fluorescein (1 mg/mL) (Merck Millipore) to the apical
compartment. Transport of the radiolabeled compounds was investigated alone and in the presence of 10 mM unlabeled compound
or 1000 nM exendin9-39. The cells were placed on a temperature-controlled shaking table at 37 C for 90 rounds per minute. Samples
were taken from the receiver compartment after 30, 60, 120, 180 and 240 min and from the donor compartment after 240 min (to
confirm mass balance). After 240 min, the experiments were terminated and permeable supports were washed and collected as
described above. Samples were transferred to Ultima Gold scintillation fluid (Perkin-Elmer) and treated as described above.
Uptake studies
On day 6 (BBB vessel model) or day 7 (BBB tanycytes model), on permeable-transwell supports, the culture medium was replaced
with Hank’s balanced salt solution with calcium and magnesium (Gibco) supplemented with 0.1% ovalbumin (Sigma-Aldrich),

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0.005% Tween 20 and 10 mM HEPES (Gibco) (pH set to 7.4) (uptake buffer). The cells were incubated 15 min at 37 C with in clean
uptake buffer or uptake buffer +1000 nM exendin9-39. For uptake of fluorescently-labeled compounds (Alexa 594-liraglutide or Alexa
488-GLP-1), the cells were added either clean uptake buffer (control) or uptake buffer + fluorescently-labeled compound (final con-
centration of 100 nM) to the apical compartment and incubated 1 h at 37 C with shaking. For uptake of 125I-liraglutide, the cells were
added to uptake buffer containing 0.7 nM 125I-liraglutide (1 mCi/mL) with varying amounts of unlabeled liraglutide (10–10,000 nM) in
the apical compartment and uptake buffer in the basolateral compartment. The cells were incubated 60 min at 37 C with shaking.
Receiver and donor samples were withdrawn, the uptake buffer was aspirated and the cells were washed three times in ice-cold
uptake buffer. The supports from the uptake studies with fluorescently-labeled compounds were fixed in 4% paraformaldehyde, per-
meabilized in 0.1% Triton X-100 and blocked in PBS +2% BSA (Sigma-Aldrich). The cells were subsequently incubated with Hoechst
(1 mg/mL) and visualized using confocal microscopy as above. The supports from the isotope experiments were either cut out to
quantify uptake or added fresh uptake buffer to measure release. For the release studies, the cells were incubated up to 1 h in uptake
buffer. The permeable supports were put into a picocount scintillation plate (PicoPlate-96, Perkin Elmer) and counted in a (Perkin
Elmer). Counts per minute were converted to molar quantities using a standard curve of 125I-liraglutide in uptake buffer run in parallel.
The amounts were standardized relative to the amounts present in the individual donor solutions to obtain the amount of compound
taken up relatively to the amount added.
Immunohistochemistry
In vitro models
Freshly isolated mouse capillary fragments, tanycyte- and endothelial cell cultures were fixed in methanol:acetone (1:1) for 1 min
at 20 C, washed three times in PBS +2% BSA and subsequently blocked for 30 min in the same buffer. Samples were incubated
with primary antibodies, rabbit claudin-5 (1:200, 34–1600, Zymed, Thermo Fisher; RRID: AB_2533157), rabbit Phospho-CREB
(1:400; 9198s, Cell Signaling, Danvers, MA, USA; RRID: AB_2561044), rabbit GFAP (1:2000, Z0334, DakoCytomation,
DakoCytomation, Glostrup, Denmark; RRID: AB_10013382), mouse GLP1-R (1:1000, mouse anti-mouse MAB 7f38 Novo-Nordisk;
RRID: AB_2618101), chicken vimentin (1:2000, AB5733 Merck Millipore, Burlington, MA, USA; RRID: AB_11212377), rabbit von
Willebrand’s Factor (1:200, F3520, Sigma-Aldrich; RRID: AB_259543), rabbit PECAM-1 (1:500, sc1506, Santa Cruz Biotechnology,
Dallas, TX, USA; RRID: AB_2161037), or mouse ZO-1 (1:200, 339111, Zymed, Thermo Fisher; RRID: AB_87182) in different combi-
nations over night at 4 C. Following three washing steps of 5 min, samples were incubated with Alexa 488 or Alexa 568-conjugated
secondary antibodies, either goat a-rabbit IgG (1:200; Alexa 488, A11008, RRID: AB_143165; Alexa 568 A11011, RRID: AB_143157
Invitrogen, Carlsbad, CA, USA), goat a-mouse IgG (1:200, Alexa 488, A11002, RRID: AB_1500639; Alexa 568, A11031, Invitrogen,
RRID: AB_144696) or goat a-chicken IgG (1:200, Alexa 568, A11041, Invitrogen; RRID: AB_2534098) combined with Hoechst
33258 (pentahydrate bis-benzimide, 1 mg/mL, Invitrogen, RRID: AB_2651133) for 1 h at room temperature.
In vivo models
Glass slides containing brain sections from liraglutide564-injected mice were subjected to three washes in 0.01 M PBS, an immuno-
histochemistry of vimentin (AB5733, Merck Millipore, RRID: AB_11212377) was performed as described before (Langlet et al.,
2013b), followed by an incubation with Alexa 488-conjugated secondary antibody goat anti-chicken IgG (1:400, A11039, Invitrogen,
RRID: AB_142924).
Double immunohistochemistry pCREB-vimentin
Ten minutes after saline or liraglutide IV administration (90 nmol/kg), 30 mm free-floating sections from perfused animals were incu-
bated with both chicken anti vimentin (AB5733, Merck Millipore, RRID: AB_11212377) and rabbit anti phospho-CREB (Hagiwara
et al., 1993) (provided by Marc R. Montminy, Salk Institute, La Jolla, CA, USA) as previously described (Sanchez et al., 2010).
c-Fos immunohistochemistry
One hour or 10 min after saline or liraglutide IV administration (90 nmol/kg), control and iBot mice were perfused and brains were
processed as described above. 30 mm free-floating sections were blocked in an incubation solution of PBS, 0.25% BSA (BSA,
A9418, Sigma-Aldrich), and 0.3% Triton X-100 (T8787, Sigma-Aldrich) with 4% normal goat serum (NGS, D9663, Sigma-Aldrich,
RRID: AB_2810235) for 2h at room temperature (RT, 20–25 C). Then, sections were incubated with rabbit c-Fos antibody (1:500,
226008, Synaptic Systems, Gottingen, Germany, RRID: AB_2231974) in the same blocking buffer for 48h in agitation at 4 C. After
PBS rinses, sections were incubated with biotinylated-goat anti rabbit antibody (1:400, 111-065-144, Jackson ImmunoResearch,
West Grove, PA, USA, RRID: AB_2337965) for 2h at RT. After PBS washes, immunoreactivity was revealed using with Alexa-Fluor
568-streptavidin (1:400, S11226, Invitrogen, RRID: AB_2315774) for 90 min. After Hoechst 33258 (pentahydrate bis-benzimide,
1 mg/mL, Invitrogen, RRID: AB_2651133) incubation for nuclei visualization, sections were mounted on glass slides and covered
with Mowiol-containing mounting medium (Calbiochem, Merck Millipore).
GFP immunohistochemistry
After the microdialysis experiment in control and GLP1RtanKO mice, brains were collected and post-fixed in 4% PFA for 24 h and then
kept in 0.01M PBS-0.05% Na azide until its processing. Brain section of 80 mm were cut using vibratome and free-floating sections
were blocked in an incubation solution of PBS, 0.25% BSA (BSA, A9418, Sigma-Aldrich), and 0.3% Triton X-100 (T8787, Sigma-Al-
drich) with 4% normal goat serum (D9663, Sigma-Aldrich, RRID: AB_2810235) for 2h at room temperature (RT, 20–25 C). Then, sec-
tions were incubated with rabbit GFP antibody (1:500, A11122, Thermo Fisher Scientific Cat# A-11122, RRID: AB_221569) in the
same blocking buffer for 48h under smooth agitation at 4 C. After PBS rinses, immunoreactivity was revealed with Alexa
488-conjugated secondary antibody goat anti-chicken IgG (1:400, A11039, Invitrogen, RRID: AB_142924) for 90 min. After Hoechst

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33258 (pentahydrate bis-benzimide, 1 mg/mL, Invitrogen, RRID: AB_2651133) incubation for nuclei visualization, sections were
mounted on slides and covered with Mowiol containing mounting medium (Calbiochem, Merck Millipore).
RNAscope fluorescent in situ hybridization

Mouse GLP1R mRNA fluorescent in situ hybridization was performed on frozen brain sections in the mediobasal hypothalamus con-
trol and GLP1RtanKO mice with the RNAscope Multiplex Fluorescent Kit v2 according to the manufacturer’s protocol (Advanced Cell
Diagnostics, Newark, CA, USA). Specific probes were used to detect GLP1R (Mm-Glp1r-C3, NM_021332.2) mRNA. Hybridization
with a probe against the Bacillus subtilis dihydrodipicolinate reductase (dapB) gene (320871) was used as negative control. Following
GLP1R FISH, a vimentin immunostaining was done using chicken anti-Vimentin antibody (1:500 Millipore Cat# AB1620, RRID:
AB_90774), revealed with Goat anti-chicken 647 (1:500; Thermo Fisher Scientific Cat# A-21449, RRID: AB_2535).
Image analysis
The capillaries or cultured endothelial cells were mounted on coverslips and visualized with a Zeiss LSM 510 laser confocal micro-
scope (Carl Zeiss, Jena, Germany, RRID: SCR_018062). Images from brain sections were taken with a Zeiss 20x objective (N.A. 0.8)
mounted on an Axio Imager Z2 light microscope (Zeiss, RRID: SCR_018856) or with Zeiss 10x objective mounted on an Axioscan
microscope (Zeiss). For FISH experiments and immunofluorescent staining of Vimentin, acquisition of images was performed using
an inverted confocal microscope (LSM 710, Zeiss, Jena, Germany) and high magnification photomicrographs were acquired with a
63x objective (NA 1.4) using the Airyscan detector (Zeiss). ImageJ (National Institutes of Health, Bethesda, MD, RRID: SCR_003070)
and Photoshop CC (Adobe Systems, San Jose, CA, RRID: SCR_014199) were used to process, quantify, adjust, and merge the pho-
tomontages. Image analyses were performed in a blinded manner. Figures were prepared using Adobe Photoshop CC and Adobe
Illustrator CC (Adobe Systems, San Jose, CA, RRID: SCR_010279).
QUANTIFICATION AND STATISTICAL ANALYSIS
No statistical method was used to determine sample size. Data are expressed as means ± SEM. To test whether the results followed a
Gaussian distribution, a normality test was performed (Kolgomorov-Smirnov test for n = five to seven, Shapiro-Wilk test for n R 7).
Results between groups were analyzed using one-way and two-way ANOVA, with Tukey’s or Fisher’s LSD post hoc tests were used
for multiple comparisons. Student’s t test between control and treatment groups in cases in which statistical significance was estab-
lished. Differences between two groups were determined using paired or unpaired Mann-Whitney for not Gaussian distributions or
one or two-tailed Student’s t tests for Gaussian ones. Statistical analyzes was performed with GraphPad PRISM 8.0 (version 8.4.2;
GraphPad Software, La Jolla, California, USA, RRID: SCR_002798). The threshold for significance was p < 0.05. All the statistical
parameters can be found in the figures and figure legends.

Cell Metabolism, Volume 34
Supplemental information
Tanycytes control hypothalamic liraglutide

uptake and its anti-obesity actions
Monica Imbernon, Chiara Saponaro, Hans Christian Cederberg Helms, Manon
Duquenne, Daniela Fernandois, Eleonora Deligia, Raphael G.P. Denis, Daniela Herrera
Moro Chao, Sowmyalakshmi Rasika, Bart Staels, François Pattou, Frank W.
Pfrieger, Birger Brodin, Serge Luquet, Caroline Bonner, and Vincent Prevot
Supplementary Figure 1. Characterization and validation of in vitro blood-brain barrier models
(Related to Fig. 1). (a) Illustration of the bovine endothelial cell/rat astrocyte BBB contact co-culture
model with endothelial cells cultured on transwell permeable supports and astrocytes covering the lower
surface of the support. (b) Photomicrograph showing bovine endothelial cells in co-culture with rat
astrocytes after immunocytochemical labeling with antibodies against the gap junction markers claudin-
5 (green) and ZO-1 (red). Yellow color indicates co-localization of the markers. Scale bar 30 µm. (c)
Trans-endothelial electrical resistance (TEER) was measured to validate the junctional integrity of the
in vitro BBB model. Each data point represents the mean of several permeable supports (technical
replicates) from an individual preparation. Line and whiskers indicate mean and SEM across biological
replicates (n = 13). (d) Transcellular permeation of 125I-labelled GLP-1 (0.5 nM) and liraglutide (0.7 nM)
measured in the BBB model and compared to the permeation of 4 kDa FITC-dextran (FD-4) and sodium
1
fluorescein. Bars show means ± SEM (n = 6, 3, 3, 3, 3, 5, 3 wells from 2 independent cultures). (e) Rat
brain cortical endothelial cells on a transwell insert (I), bright-field photomicrograph of endothelial cell
cultures from rat cortical capillaries (II), immunocytochemical labeling for the endothelial marker
PECAM-1 (green), vimentin (red), a marker for tanycytes in the hypothalamus, von Willebrand’s factor
(green), which also labels endothelial cells, and GLP1R (green) (III-V); scale bar: 30 µm. (f) Tanycytes
cultured on transwell permeable supports (I), bright-field image of tanycytic cultures from median
eminence explants (II), immunofluorescence labeling for vimentin (red), GFAP (green) and GLP1R
(green) (III-IV) scale bar: 30 µm. (g) Diagram and gating strategy for sorting Tomato-positive putative
tanycytes following TAT-Cre infusion into the third ventricle (3V) of tdTomatoloxP-STOP-loxP mice and
GLP1R mRNA expression in tanycytes isolated by FACS from tdTomato mice (n = 5).
2
Supplementary Figure 2. Liraglutide564 is taken up by tanycytes, which express GLP1R in vivo,
in a GLP1R-dependent manner and promotes CREB phosphorylation in vitro (Related to Fig. 1
and Fig. 2). (a) Photomicrographs of cultured tanycytes following immunocytochemical staining for
pCREB (red) after treatment with control uptake buffer (left panel), 100 nM liraglutide564 (middle panel)
and 1000 nM exendin 9-39 and subsequently with 100 nM liraglutide564; Scale bar: 30 µm. (b) Diagram
and gating strategy for sorting EGFP-positive putative tanycytes following TAT-Cre infusion into the third
ventricle (3V) of BoNTB-EGFPloxP-STOP-loxP mice (iBot mice). (c) EGFP-positive cells (green) express
DARPP-32 mRNA (tanycytic marker), but not NPY mRNA (neuronal marker) or MECA32 mRNA
(fenestrated endothelial cell marker). Expression in EGFP-negative cells has been normalized to 1
(dotted line). Mann-Whitney U test, ** p=0.0079, n = 5 mice. (d) GLP1R mRNA expression in EGFP-
negative (black bar) and EGFP-positive (green bar) cells isolated from iBot mice; data are expressed
as means ± SEM. ** Mann-Whitney test, p=0.0079, n = 6 mice.
3
Supplementary Figure 3. Liraglutide-induced c-Fos activation is abolished in the hypothalamus
of iBot mice selectively expressing botulinum toxin in tanycytes (Related to Fig. 2). (a-d)
Representative photomicrographs of c-Fos immunohistochemistry in the hypothalamus of control (a, b)
and iBot (c, d) mice 10 min after intravenous injection of saline (left panels) or liraglutide (90 nmol/Kg,
right panels). AHN: anterior hypothalamic nucleus; ARH: arcuate nucleus of the hypothalamus; DMH:
dorsomedial hypothalamic nucleus; LHA: lateral hypothalamic nucleus; PVH: paraventricular nucleus of
the hypothalamus; TUB: tuberal nucleus; ZI: zona incerta. Scale bar 200 µm. (e) Quantification of the
number of c-Fos positive cells in mouse hypothalamic sections 10 minutes after either intravenous saline
(black, grey) or liraglutide (90nmol/kg) injection (green, blue) in control (black and green) and iBot mice
(grey and blue); n= 4 animals per group; 6 to 7 sections per animal. AHN: two-way ANOVA, genotype:
5
F(1, 12)= 3.09, p = 0.104; treatment: F(1, 12)= 7.55, p = 0.017; interaction: F(1, 12)= 0.276, p = 0.276. Fisher’s
LSD post hoc test, control saline vs. control liraglutide, p = 0.0176. PVH: two-way ANOVA, genotype:
F(1, 12)= 2.92, p = 0.113; treatment: F(1, 12)= 2.39, p = 0.147; interaction: F(1, 12)= 2.03, p = 0.1794. Fisher’s
LSD post hoc test, control saline vs. control liraglutide, p = 0.05; control liraglutide vs. iBot liraglutide, p
= 0.046. LHA: two-way ANOVA, genotype: F(1, 10)= 11.3, p = 0.007; treatment: F(1, 12)= 21.31, p = 0.001;
interaction: F(1, 10)= 2.143, p = 0.174. Fisher’s LSD post hoc test, control saline vs. control liraglutide, p
= 0.002; iBot saline vs. iBot liraglutide, p = 0.0002; control liraglutide vs. iBot liraglutide, p = 0.006. Data
are expressed as means ± SEM. *p < 0.05; *** p < 0.001, control saline vs. control liraglutide; #### p <
0.0001, control liraglutide vs. iBot liraglutide.
6
Supplementary Figure 4 (Related to Fig. 3 and Fig. 4). Effect of liraglutide on energy expenditure in
iBot mice. ( a ) Energy expenditure in iBot and control mice after 3 days of intraperitoneal treatment with
saline (baseline, grey and black respectively) or liraglutide (blue and green respectively, 0.1 mg/Kg/day).
Line plot shows mean energy expenditure at different times 3 days after liraglutide treatment compared
to baseline, and AUC. AUC paired two-tailed t-test, t(7) = 5.73, p = 0.0007. Dotted arrow indicates saline
or liraglutide administration (n = 9, 8, 8, 8 mice). Two-way ANOVA with Tukey’s post hoc test was
performed on AUC (control saline vs. control liraglutide, p < 0.0001; control liraglutide vs. iBot liraglutide,
p < 0.0001) (n = 9, 8, 7, 7 mice). Data are expressed as means ± SEM *** p <0.001. (b) Selective
expression of GFP in tanycytic cell bodies and processes in animals transduced with control and shRNA
viruses. (c) Expression of GPR50 mRNA (tanycytic marker) but not POMC mRNA (neuronal marker) by
7
GFP-positive cells (green), and vice versa in GFP-negative cells (black) FACS-isolated from mice
transduced with control-GFP or shRNA-GLP1R-GFP viral vectors. Unpaired one-tailed t-test (GPR50,
positive vs. negative, t(22) = 7.03, p < 0.0001, n = 12 mice; POMC, positive vs. negative, t(18) = 6.49, p <
0.0001, n = 10 mice). (d) Correlation between optical density (O.D.) with total GLP1 (black line) or
liraglutide (green line) amounts detected with the total GLP1 ELISA kit.

Imbernon Tanycytes Control Hyphotalamic Liranglutide Utake and Its Anti Obesity Actions

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Imbernon Tanycytes Control Hyphotalamic Liranglutide Utake and Its Anti Obesity Actions

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Short Article

Tanycytes control hypothalamic liraglutide uptake

d Endothelial cells of the blood-brain barrier do not

d Tanycytic liraglutide transport is essential to activate target

d Blocking tanycytic transport or GLP1R impairs the anti-

Imbernon et al., 2022, Cell Metabolism 34, 1054–1063

*Correspondence: caroline.bonner@univ-lille.fr (C.B.), vincent.prevot@inserm.fr (V.P.)

INTRODUCTION carries an additional albumin-binding fatty-acid side chain

Cell Metabolism 34, 1054–1063, July 5, 2022 1055

1056 Cell Metabolism 34, 1054–1063, July 5, 2022

Cell Metabolism 34, 1054–1063, July 5, 2022 1057

1058 Cell Metabolism 34, 1054–1063, July 5, 2022

Cell Metabolism 34, 1054–1063, July 5, 2022 1059

1060 Cell Metabolism 34, 1054–1063, July 5, 2022

d KEY RESOURCES TABLE

Cell Metabolism 34, 1054–1063, July 5, 2022 1061

1062 Cell Metabolism 34, 1054–1063, July 5, 2022

Cell Metabolism 34, 1054–1063, July 5, 2022 1063

KEY RESOURCES TABLE

REAGENT or RESOURCE SOURCE IDENTIFIER

e1 Cell Metabolism 34, 1054–1063.e1–e7, July 5, 2022

Cell Metabolism 34, 1054–1063.e1–e7, July 5, 2022 e2

Data and code availability

EXPERIMENTAL MODEL AND SUBJECT DETAILS

Stereotaxic TAT-Cre delivery

e3 Cell Metabolism 34, 1054–1063.e1–e7, July 5, 2022

GLP1R knockdown in tanycytes

In vivo brain microdialysis

Intrajugular administration of liraglutide

Indirect calorimetry study

Isolation of tanycytes by FACS

Cell Metabolism 34, 1054–1063.e1–e7, July 5, 2022 e4

Quantitative RT-PCR analyses

Establishment of in vitro BBB and tanycyte-models

TEER and transport assessments

e5 Cell Metabolism 34, 1054–1063.e1–e7, July 5, 2022

Cell Metabolism 34, 1054–1063.e1–e7, July 5, 2022 e6

RNAscope fluorescent in situ hybridization

QUANTIFICATION AND STATISTICAL ANALYSIS

e7 Cell Metabolism 34, 1054–1063.e1–e7, July 5, 2022

Tanycytes control hypothalamic liraglutide

in a GLP1R-dependent manner and promotes CREB phosphorylation in vitro (Related to Fig. 1

as means ± SEM. ** Mann-Whitney test, p=0.0079, n = 6 mice.

Representative photomicrographs of c-Fos immunohistochemistry in the hypothalamus of control (a, b)

0.0001, control liraglutide vs. iBot liraglutide.

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