ORIGINAL RESEARCH
published: 11 September 2018
doi: 10.3389/fendo.2018.00523
Frontiers in Endocrinology | www.frontiersin.org 1September 2018 | Volume 9 | Article 523
Edited by:
Ichiro Maruyama,
Okinawa Institute of Science and
Technology, Japan
Reviewed by:
Rocco Bruno,
Independent researcher, Matera, Italy
Marco António Campinho,
Centro de Ciências do Mar (CCMAR),
Portugal
Tania M. Ortiga-Carvalho,
Universidade Federal do Rio de
Janeiro, Brazil
*Correspondence:
Noushafarin Khajavi
Specialty section:
This article was submitted to
Molecular and Structural
Endocrinology,
a section of the journal
Frontiers in Endocrinology
Received: 18 June 2018
Accepted: 21 August 2018
Published: 11 September 2018
Citation:
Bräunig J, Mergler S, Jyrch S,
Hoefig CS, Rosowski M, Mittag J,
Biebermann H and Khajavi N (2018)
3-Iodothyronamine Activates a Set of
Membrane Proteins in Murine
Hypothalamic Cell Lines.
Front. Endocrinol. 9:523.
doi: 10.3389/fendo.2018.00523
3-Iodothyronamine Activates a Set of
Membrane Proteins in Murine
Hypothalamic Cell Lines
Julia Bräunig1,2, Stefan Mergler3, Sabine Jyrch1,2, Carolin S. Hoefig4,5, Mark Rosowski6,
Jens Mittag5,7, Heike Biebermann1,2 and Noushafarin Khajavi1,2*
1Charité – Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and
Berlin Institute of Health, Berlin, Germany, 2Institute of Experimental Pediatric Endocrinology, Berlin, Germany, 3Klinik für
Augenheilkunde, Charité – Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin, Humboldt-Universität zu
Berlin, and Berlin Institute of Health, Berlin, Germany, 4Institute of Experimental Endocrinology, Charité – Universitätsmedizin
Berlin, Corporate Member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Berlin,
Germany, 5Department of Cell & Molecular Biology, Karolinska Instituet, Stockholm, Sweden, 6Department Medical
Biotechnology, Institute of Biotechnology, Technical University of Berlin, Berlin, Germany, 7University of Lübeck – Center of
Brain Behavior and Metabolism, Lübeck, Germany
3-Iodothyronamine (3-T1AM) is an endogenous thyroid hormone metabolite. The
profound pharmacological effects of 3-T1AM on energy metabolism and thermal
homeostasis have raised interest to elucidate its signaling properties in tissues
that pertain to metabolic regulation and thermogenesis. Previous studies identified
G protein-coupled receptors (GPCRs) and transient receptor potential channels
(TRPs) as targets of 3-T1AM in different cell types. These two superfamilies of
membrane proteins are largely expressed in tissue which influences energy balance
and metabolism. As the first indication that 3-T1AM virtually modulates the function
of the neurons in hypothalamus, we observed that intraperitoneal administration
of 50 mg/kg bodyweight of 3-T1AM significantly increased the c-FOS activation
in the paraventricular nucleus (PVN) of C57BL/6 mice. To elucidate the underlying
mechanism behind this 3-T1AM-induced signalosome, we used three different murine
hypothalamic cell lines, which are all known to express PVN markers, GT1-7,
mHypoE-N39 (N39) and mHypoE-N41 (N41). Various aminergic GPCRs, which are
the known targets of 3-T1AM, as well as numerous members of TRP channel
superfamily, are expressed in these cell lines. Effects of 3-T1AM on activation of
GPCRs were tested for the two major signaling pathways, the action of Gαs/adenylyl
cyclase and Gi/o. Here, we demonstrated that this thyroid hormone metabolite has
no significant effect on Gi/osignaling and only a minor effect on the Gαs/adenylyl
cyclase pathway, despite the expression of known GPCR targets of 3-T1AM. Next,
to test for other potential mechanisms involved in 3-T1AM-induced c-FOS activation
in PVN, we evaluated the effect of 3-T1AM on the intracellular Ca2+concentration
and whole-cell currents. The fluorescence-optic measurements showed a significant
increase of intracellular Ca2+concentration in the three cell lines in the presence of
10 µM 3-T1AM. Furthermore, this thyroid hormone metabolite led to an increase of
whole-cell currents in N41 cells. Interestingly, the TRPM8 selective inhibitor (10 µM
AMTB) reduced the 3-T1AM stimulatory effects on cytosolic Ca2+and whole-cell
Bräunig et al. 3-Iodothyronamine-Induced Hypothalamic Signaling Network
currents. Our results suggest that the profound pharmacological effects of 3-T1AM
on selected brain nuclei of murine hypothalamus, which are known to be involved in
energy metabolism and thermoregulation, might be partially attributable to TRP channel
activation in hypothalamic cells.
Keywords: 3-T1AM, signaling, hypothalamus, GPCR, TRP Channel
INTRODUCTION
3-iodothyronamine (3-T1AM) is a decarboxylated and
deiodinated derivative of thyroid hormones (1,2). Although
several studies detected 3-T1AM in human blood (3,4), the
mechanism of physiological action of this compound in the
human body remains undefined. Administration of 3-T1AM
in rodents blocks the hypothalamic—pituitary—thyroid axis
and results in concentration-dependent reversible effects on
body temperature, energy metabolism, cardiac and neurological
functions (1,5). Previous observations in rodents demonstrated
the accumulation of 3-T1AM in different tissues such as kidney,
liver, muscles, and brain (6). In mice, after administration of
a radioisotope labeled [125I]-3-T1AM, 125I was detected in the
brain (6). In rats, site-directed injections of 3-T1AM into the
locus coeruleus elicits significant neuronal firing rate changes
in the selected brain nuclei such as the paraventricular nucleus
(PVN) of the hypothalamus (7). Interestingly, the target areas
of 3-T1AM in the brain nuclei are mostly involved in energy
homeostasis, arousal, and memory retrieval (8–11). Presumably,
different effects of 3-T1AM, such as anapyrexia and food
consumption might be centrally mediated via the hypothalamus
(12–14). Due to the profound pharmacological effects of 3-
T1AM and its accumulation in the selected tissue, numerous
studies over the last years have been devoted to investigating the
signaling property of this thyroid hormone metabolite.
The first target of 3-T1AM is the trace amine associated
receptor 1 (TAAR1), a trace amine-activated G protein-coupled
receptor (GPCR) (1). 3-T1AM induces Gαs/adenylyl cyclase
signaling in rodent TAAR1 and human TAAR1-transfected cells
(1). Additionally, different studies described several other GPCRs
as 3-T1AM targets, mainly in vitro and in overexpressing systems.
These GPCRs belong to the group of aminergic GPCRs (15)
such as the α-2A-adrenergic receptor (ADRA2A (16), the β2-
adrenergic receptor (ADRB2) (17), the muscarinergic receptor
3 (CHRM3) (18), or the serotonin receptor 1b (5-HT1b)
(19). Moreover, 3-T1AM modulates calcium and potassium
homeostasis through an intracellular calcium channel, known as
“ryanodine receptor” in adult rat cardiomyocytes (20).
Recent studies identified non-selective cation channels such as
the transient receptor potential channel melastatin 8 (TRPM8)
and the transient receptor potential vanilloid 1 (TRPV1)
as novel targets of 3-T1AM (21–23). Classically, TRPM8 is
known as a cold and menthol receptor and is a temperature-
sensitive receptor in excitable cells (24). Its activation induces a
depolarization of the cell membrane leading to action potentials.
The same function principle applies to TRPV1, which is known
as a heat- and capsacin receptor (25). Together, these properties
implicate these TRPs as possible transducers of cold or warm
stimuli not only within the hypothalamus (26), but also in
keratintocytes of human skin (27) and neurons on human corneal
nerve fibers (28,29).
Different studies demonstrated that TRPs are the major
downstream effectors of GPCRs and the signaling cascades that
emanate from the activation of GPCR evoke TRP channel activity
(30,31). There is a wide distribution of TRPs in tissues that
influence energy homeostasis and thermoregulation. Expression
of TRPs in various tissues such as hypothalamus, peripheral
sensory neurons, gastrointestinal tract, liver, adipocytes, and
ocular tissues strongly suggest the possible role these ion
channels play in energy balance and metabolism as well as
thermoregulation (32–37). Modulation of TRPs via 3-T1AM
raises the question of what could be the 3-T1AM-induced
signalosome and whether there is a link between stimulatory
effects of 3-T1AM in tissues that pertain to metabolic- and/or
thermo-regulation and TRPs.
Here, we identified the stimulatory effect of 3-T1AM in
murine hypothalamic nuclei and explored the underlying
mechanism behind this effect in murine hypothalamic cell
lines. The results of this study show a stimulatory effect of 3-
T1AM on Ca2+mobilization and whole-cell currents in murine
hypothalamic cells and that this effect is associated with TRPM8
activation.
METHODS
Mice Experiments
Immunohistochemistry
In collaboration with the Karolinska Institute, Sweden, C57BL/6J
mice (4 in each group) were i.p. injected with 50 mg/kg body
weight 3-T1AM solved in 60% DMSO and 40% PBS, control
mice with 60% DMSO and 40% PBS (volume of injection was
5µl/g body weight). After 60 min, animals were transcardially
perfused with PBS and 10% formalin (European Community
Council Directives (86/609/EEC) and approved by Stockholm’s
Norra Djurförsöksetiska Nämnd). Fixated murine brains were
successively incubated in 10, 20, and 30% sucrose solution
over several days. Brains were cut at a cryotom into 30 µm
slices and placed in a 48 well plate filled with PBS. Slices were
blocked with a blocking buffer (TBS, 0.25% gelatin from porcine
skin and 0.5% triton X100) for 2 h, subsequently incubated
with a c-FOS antibody, rabbit anti mouse (1:200; Santa Cruz
Biotechnology, Santa Cruz, CA, USA) over night at 4◦C and
finally with an Alexa Fluor 549 antibody, goat anti rabbit
(1:200; Jackson ImmunoResearch) for 2 h at room temperature.
Between each step, the slides were washed 3 ×1 min with
TBS (tris-buffered saline). The brain slices were placed on
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Bräunig et al. 3-Iodothyronamine-Induced Hypothalamic Signaling Network
glass slides and mounted with VectaShield containing DAPI
(Vector Laboratories, Burlingame, CA, USA). Pictures were
taken with a Keyence BZ-9000 microscope (20×magnification)
and optimized with the ImageJ software. C-FOS positive cells
were counted with the ImageJ software using two brain slides of
each animal for each brain loci.
Cell Culture and mRNA Isolation
GT1-7 (mouse hypothalamic gonadotropin-releasing-hormone
neuronal cell line) were purchased from MERK, (38). mHypoE-
N39 (N39) and mHypoE-N41 (N41), both embryonic mouse
hypothalamic cell lines, were acquired from Cedarlane,
established by Belsham et al. (39). A screening profile of neuronal
markers of all three cell lines can be viewed here (https://www.
cedarlanelabs.com/Products/Listing/Hypothalamic). The cells
were cultured in Dulbecco’s modified Eagle’s medium (DMEM,
Biochrom GmbH, Berlin, Germany), supplemented with 10%
fetal calf serum (FCS) and 1% penicillium/streptavidin at 37◦C
in humidified air containing 5% CO2.
Cells were seeded in T75 flask and grown to 80% confluence.
Cells were harvested in three different passages, spanning from
passage three to passage six. Total mRNA was isolated by
chloroform/phenol extraction, a DNase digestion was performed
and samples were stored at −80◦C.
Quantitative PCR to Determine GPCR and
TRP Channel Expression Profiles
A quantitative PCR (qPCR) was performed to determine the
expression level of several GPCRs and TRPs. QPCR primers
including their efficiency are listed in Supplemental Table 1.
Pgk1 was chosen as the reference gene, as previously
recommended (40). First, mRNA was transcribed into cDNA
by the Ominscript RT kit (Qiagen) using random hexameres
(Applied Biosystems) and Oligo dTs (Promega, Madison, USA).
Absolute QPCR Mix, SYBR Green, no Rox (Thermo Scientific,
Germany) was used for qPCR on a Stratagene Mx3000P System
using 100 nM per primer. PCR reaction underwent an initial
cycle at 95◦C for 15 min followed by 42 cylces at 95◦C for 15s,
primer specific annealing step 60◦C for 30 s and a elongation
step 72◦C for 45 s, and elongation at 72◦C for 7 min and
finally temperature holding at 4◦C. Melting curve analysis was
performed to confirm the specificity of the PCR reaction. Data
was processed using the 1ct method. We used the slope of a
standard curve to determine the amplification efficiency for each
primer pair (efficiency =10−1/slope). Each of the three passages
of every cell line was measured in duplicates together with a
sample without reverse transcription to exclude genomic DNA
contamination.
Gαsand Gi/oSignaling of Endogenous
Expressed GPCRs in Hypothalamic Cell
Lines
Gαsand Gi/osignaling were determined by measuring cAMP
accumulation using the AlphaScreen technology (PerkinElmer
Life Science, Boston, MA, USA) as previously described
(15). Cells were cultured in poly-L-lysine (Biochrom GmbH,
Berlin, Germany) coated 96-well plates (1 ×10−4cells
/ well). Seventy-two hours after seeding, stimulation was
performed by means of using a stimulation buffer (138 mM
NaCl, 6 mM KCl, 1 mM [MgCl∗
26H2O], 5.5 mM glucose,
20 mM HEPES, 1 mM [CaCl∗
22H2O], 1 mM IBMX). For Gαs
signaling, cells were incubated for 45 min with either 3-T1AM
(Santa Cruz Biotechnology, Dallas, TX, USA), serotonin (5-
HT, Sigma-Alderich, St. Louis, MO, USA), norepinephrine
(NorEpi, Sigma-Alderich, St. Louis, MO, USA), isoproterenol
(ISOP, Sigma-Alderich, St. Louis, MO, USA), or phenethylamine
(PEA, Sigma-Alderich, St. Louis, MO, USA) in a concentration
of 10 µM or only stimulation buffer to monitor the basal
cAMP content. 3-T1AM was diluted from a 10 mM stock
solution using DMSO as solvent. H2O was used as solvent
for serotonin and norepinephrin and PBS with 0.1% BSA was
used as the solvent for isoproterenol and phenethylamine. For
Gi/opathway examination, cells were additionally stimulated
with 50 µM forskolin (FSK, AppliChem GmbH, Darmstadt,
Germany) to activate the adenylyl cyclase for a total of
45 min. Afterwards, cells were lysed at 4◦C on a shaking
platform. Intracellular cAMP accumulation was determined by
a competitive immunoassay based on the AlphaScreen assay kit
according to the manufacturer’s instructions and measured using
a Berthold Microplate Reader (Berthold Technologies GmbH &
Co. KG, Bad Wildbad, Germany). Cyclic AMP concentrations
were normalized to protein contents, which was measured
with the Pierce BCA Protein Assay Kit (Thermo Scientific,
Germany).
Determination of Intracellular Ca2+
Concentration
To monitor time-dependent changes in intracellular free Ca2+
levels ([Ca2+]i) in single-cells, cells were pre-incubated with
culture medium containing fura-2/AM (2 µM) for ∼30 min at
37◦C. Loading was stopped with a Ringer-like (control) solution
containing: 150 mM NaCl, 6 mM CsCl, 1 mM MgCl2, 10 mM
glucose, 10 mM HEPES, and 1.5 mM CaCl2at pH 7.4. Where a
blocker was used, pre-incubation was performed 30 min before
the measurement. Fluorescence measurements were performed
on the stage of an invert microscope (Olympus BW50WI)
and a camera (Olympus XM-10) in connection with a LED-
Hub (Omikron, Rodgau-Dudenhoven, Germany). Fura-2/AM
fluorescence was excited at 340 and 380 nm alternatingly and
emission was detected from small cell clusters every 4 s at 510 nm.
Results are shown as mean traces of f340nm/f380nm ±SEM. Drugs
were dissolved in dimethyl sulfoxide (DMSO) to obtain a stock
solution and diluted in Ringer-like solution to obtain a working
concentration which did not exceed 0.1%. For image acquisition
and data evaluation, the life science imaging software cellSens
was used (Olympus, Hamburg, Germany).
Planar Patch-Clamp Recordings
For electrophysiological recordings, the semi-automated planar
patch-clamp technique was used as previously described (41).
Whole-cell currents were evaluated in conjunction with an
EPC10 amplifier and PatchMaster acquisition software (HEKA,
Lambrecht, Germany) as well as PatchControl software (Nanion,
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Bräunig et al. 3-Iodothyronamine-Induced Hypothalamic Signaling Network
Munich, Germany). For recording, 5 µl of an internal-like
solution was applied to the internal side of the microchip. The
internal solution contained: 50 mM CsCl, 10 mM NaCl, 2 mM
MgCl2, 60 mM CsF, 20 mM EGTA, and 10 mM HEPES, pH
7.2 and osmolarity 288 mOsM. Cs in the internal solution
blocks potassium channel activity. A single cell suspension was
added to an external solution of the following composition:
140 mM NaCl, 4 mM KCl, 1 mM MgCl2, 2 mM CaCl2F, 5 mM
D-glucose monohydrate, and 10 mM HEPES, pH 7.4 and
osmolarity 298 mOsM. Whole-cell currents were recorded using
a ramp protocol ranging between −60 to +130 mV for 500
milliseconds. The mean membrane capacitance of N41 cells
was 9 pF ±1 pF (n=10). Mean access resistance was
15 ±1 M(n=10). The holding potential (HP) was set
to 0 mV in order to eliminate any possible contribution of
VDCCs or sodium channels. All plots were generated with
SigmaPlot software version 12.5 (Systat, San Jose, California,
USA).
Data Evaluation and Statistics
GraphPad Prism 6.0 (GraphPad software, San Diego, Calif., USA)
was chosen for visualization and data analysis. Data are shown
as means ±SEM of independent experiments. Statistical analysis
was carried out using one-way and two-way ANOVA, followed
by Sidak correction. For Ca2+imaging, statistical significance
was determined by an unpaired t-test with Welch’s correction.
In figure legends, the number of experiments and the type of
comparison are given. Statistical significance was defined as
∗p≤0.05, ∗∗p≤0.01, ∗∗∗p≤0.001, and ∗∗∗∗p≤0.0001.
RESULTS
Intraperitoneal Injection of 3-T1AM Results
in the Activation of PVN Neurons in
C57BL/6 Mice
Previous studies demonstrated the accumulation and stimulatory
effects of 3-T1AM in selected brain nuclei such as locus coeruleus
and PVN of the hypothalamus (6,7). However, the exact role of
this thyroid hormone metabolite in the hypothalamus remains
unclear. To investigate whether 3-T1AM is capable of activating
hypothalamic neurons in these nuclei in vivo, we performed
intraperitoneal injections of 50 mg/kg bodyweight of 3-T1AM
or DMSO/PBS as control, six mice per group, and monitored
3-T1AM-induced neuron activation relative to DMSO/PBS. We
used c-FOS as the marker for neuronal activity. One hour after
3-T1AM injection, increased c-FOS staining of distinct neurons
was clearly visible in the PVN (60 ±13 c-FOS positive cells
per brain slide), while DMSO/PBS treated mice showed only
few c-FOS positive neurons (16 ±4 c-FOS positive cells per
brain slide) (Figures 1A,B). 3-T1AM had no stimulatory effect
on the medial preoptic area (MPO), the supraoptic nucleus
(SON), the dorsolmedial nucleus of the hypothalamus, the
periaqueductal gray (PAG) and the ventral tegmental segment
(VTA) (Figure 1B and Supplemental Figure 1). The c-FOS
positive cells ranged from 4 to 25 per brain slide in these brain
loci.
Expression Profile of GPCRs, the Main
3-T1AM Target, in the Murine Hypothalamic
Cell Lines
To elucidate the underlying mechanism behind the stimulatory
effect of 3-T1AM in the hypothalamus, we used three murine
hypothalamic cell lines, GT1-7, mHypoE-N39 (N39), and
mHypoE-N41 (N41). These cell lines are established models
to study neuroendocrine mechanisms and known to express
PVN-like markers (38,42,43). Here we demonstrated that in
N41 cells, 3-T1AM significantly increased the c-FOS activation
(Supplemental Figure 2). As GPCRs are the primary targets of
3-T1AM, we investigated the GPCR expression profile of the
three cell lines. Here, we showed the expression of the aminergic
receptors Taar1,5-Ht1b,Adra2a,Adrb1, and Adrb2 (Figure 2).
Furthermore, when compared against of Pgk1, Adrb1 noticeably
had the highest expression rate among these receptors in all three
cell lines with a ratio of 94 ±38 for GT1-7, 565.6 ±312 for
N39, and 1046 ±456 for N41. The second highest expression
was detected for Adrb2 with a ratio of 1.54 ±0.34 for GT1-
7, 2.77 ±1.04 for N39, and 6.06 ±2.34 for N41. The other
three receptors Taar1,5-Ht1b, and Adra2a, displayed similar
expression profiles. The mRNA content in all three cell lines
was lower than the reference gene Pgk1 (Figure 2), with ratios
between 0.37 ±0.14 for 5-Ht1b in GT1-7 and up to 0.72 ±0.08
for Taar1 in N41.
3-T1AM Induces FSK-Amplified Gαs
Signaling in the Murine Hypothalamic Cell
Lines
To investigate which pathway could contribute to 3-T1AM
actions in the hypothalamus, we tested two major signaling
cascades downstream of 3-T1AM GPCR targets, Gαsand Gi/o.
To measure endogenous Gαssignaling, we determined the cAMP
enhancement. N41 and GT1-7 cells had a higher basal cAMP
content with 3.2 ±0.36 nM cAMP/g/L protein for N41 and
3.41 ±0.26 nM cAMP/g/L protein for GT1-7, compared to N39
with 0.92 ±0.13 nM cAMP/g/L protein (Figure 3A,n=4 in
triplicates, p<0.001). In all cell lines, 3-T1AM stimulation
(10 µM) did not increase cAMP concentration compared to
the basal cAMP content (Figure 3A). Only NorEpi and ISOP
activated an endogenous Gαssignal in GT1-7 (∼1.6 fold for
NorEpi and ∼1.7 fold for ISOP), N41 (∼1.8 fold for NorEpi and
∼2.1 fold for ISOP), and N39 (∼3.5 fold for NorEpi and ∼5.9
fold for ISOP) cells (Supplemental Figure 3). 5-HT and PEA,
endogenous ligands for 5-HT1b and TAAR1, did not increase
cAMP content (Supplemental Figure 3).
To determine Gi/osignaling, cells were incubated with FSK,
an unspecific activator of the adenylyl cyclase, which increases
the cellular cAMP content. It is known that activation of
Gi/oleads to inhibition of the adenylyl cyclase and a decrease
in FSK-induced cAMP content. For all three cell lines, Gi/o
activation was not detected after stimulation with 10 µM 3-
T1AM (Figure 3B). In addition, FSK can potentiate weak Gαs
signaling. Dessauer et al. showed that FSK in the presence
of Gαshas a higher affinity to the adenylyl cyclase, yielding
in a higher cAMP accumulation (44). Here, this phenomenon
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Bräunig et al. 3-Iodothyronamine-Induced Hypothalamic Signaling Network
FIGURE 1 | Staining of c-FOS activated neurons after 1 h of i.p. injection of 50 mg/kg 3-T1AM. After intraperitoneal injection of the C57BL/6J mice with either
3-T1AM or solvent (60% DMSO/40% PBS), brains were frozen and cryosectioned and stained against c-FOS and DAPI. (A) In comparison to the control mice,
3-T1AM-treated mice showed a strong c-FOS staining in the PVN. All pictures were taken with a 20×objective. (B) c-FOS positives cells were counted in the
respective nuclei. Only 3-T1AM treated animals (n=4) showed an increase in c-FOS activity in the PVN. For statistics a two-way ANOVA was performed, followed by
a Sidak correction; ***p≤0.001.
FIGURE 2 | Expression pattern of GPCRs in N39, N41 and GT1-7. Results of a SYBR Green based qPCR. Graphs show the ratios between the reference gene Pgk1
and the GPCRs Taar1,5-Ht1b,Adra2a,Adrb1, and Adrb2. Data was pooled from (n=3) measured in duplicates. (A)Adrb1 and Adrb2 have the highest expression,
while 5-Ht1b has the lowest in GT1-7 cells. (B) In N39 cells, Adrb1 has the expression ratio compared to Pgk1, followed by Adrb2.5-Ht1b and Adra2a are at least
abundant in N39 cells. (C) Among the measured GPCRs Adrb1 has the most copies of mRNA in the transcriptome of N41 cells, once again followed by Adrb2.
However, Taar1,5-Ht1b, and Adra2a are rather equimolar to Pgk1.
FIGURE 3 | 3-T1AM induces FSK-stimulated Gαssignaling in murine hypothalamic cell lines. For Gαsand Gi/o, the cAMP content was measured via an AlphaScreen
technology. Data are pooled from four independent assays measured in triplicates (n=4). For statistics a two-way ANOVA was performed, followed by a Sidak
correction. Statistics were set to *p≤0.05, **p<0.01. (A) Cells were stimulated with stimulation buffer or 3-T1AM in a concentration of 10−5M for 45 min (n=4).
(B) Cells were co-stimulated with 50 µM FSK and either stimulation buffer or 3-T1AM (10 µM) for 45 min (n=4).
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Bräunig et al. 3-Iodothyronamine-Induced Hypothalamic Signaling Network
emerges for N39 and N41 and 3-T1AM stimulation significantly
increases cAMP content in FSK-treated cells (144.2 ±16.15%
for N39 and 160.77 ±21.17% for N41, n=4 in triplicates,
pN39 =0.0128, pN41 =0.0019). The specific ligands for
TAAR1, 5-HT1b, ADRA2A, ADRB1, and ADRB2 did not
activate Gi/osignaling (Supplemental Figure 4). Collectively, in
addition to an FSK-potentiated Gαsactivation, 3-T1AM had no
detectable influence on cAMP content in the hypothalamic cell
lines.
Gene Expression Profile of TRPs in Murine
Hypothalamic Cell Lines
The aforementioned results demonstrated that GPCR-dependent
signaling is not the sole regulator of 3-T1AM-induced effects on
hypothalamic cells. Previous studies identified several members
of TRPs as new targets for 3-T1AM (21–23). Here, we measured
the gene expression levels of the TRPM subfamily and TRPV1 in
three hypothalamic cell lines. The qPCR data show that none of
the hypothalamic cell lines expressed Trpm5 (Figure 4). Contrary
to this, in GT1-7 cells, Trpm4 was the highest expressed TRP
channel with a ratio of 159 ±89 compared to Pgk1, followed
by Trpm7 with a ratio of 11.7 ±10.1 to Pgk1.Trpv1 had a ratio
of 2.96 ±2.91 and Trpm8 of 0.03 ±0.02 to Pgk1.Trpm1 was
expressed with a ratio of 0.00075 ±0.00045 to Pgk1.Trpm6 had
the lowest expression with a ratio of 0.00000064 ±0.00000031
compared to Pgk1 (Figure 4A). In N39 cells, Trpm4 was also the
highest expressed TRP channel with a ratio of 831 ±617 to Pgk1,
followed by Trpv1 with a ratio of 79 ±38 to Pgk1.Trpm7 had a
ratio of 59 ±29 and Trpm8 a ratio of 2.19 ±0.72. Trpm1, Trpm2,
and Trpm3 expression ratios laid between a ratio of 0.029 ±0.025
to Pgk1 for Trpm2 and Trpm3 with a ratio of 0.000089 ±0.000076
to Pgk1. The mRNA content was comparable to GT1-7 cells, with
Trpm6 having the lowest expression with a ratio of 4.95 ±3.48
compared to the reference gene (Figure 4B). N41 cells exhibited
a similar expression pattern of TRP channels as GT1-7 and N39
cell lines. Trpm4 (ratio to Pgk1 759 ±674) and Trpv1 (ratio to
Pgk1 514 ±226) were the highest expressed genes, followed by
Trpm7 (ratio to Pgk1 29 ±23) and Trpm8 (24 ±18). Trpm2
and Trpm1 were lower expressed with ratios of 0.029 ±0.025
and 0.0014 ±0.0011 compared to the reference gene. Trpm3 and
Trpm6 were least expressed in the transcriptome of N41 cells with
ratios to Pgk1 of 0.000089 ±0.000076 and 0.0000022 ±0.0000013
(Figure 4C).
3-T1AM Increases Intracellular Ca2+
Concentration and Whole-Cell Currents in
Murine Hypothalamic Cell Lines
To investigate the involvement of TRPs in the 3-T1AM
stimulatory effects in Ca2+regulation, we monitored time-
dependent changes in intracellular free Ca2+levels ([Ca2+]i) in
single-cells. 3-T1AM (10 µM) increased the f340nm/f380nm ratio
from 0.70 ±0.01 to 0.77 ±0.05; (n=10) in GT1-7, from 0.70 ±
0.008 to 0.84 ±0.03 (n=15) in N39 cells and from 0.70 ±0.009
to 1.88 ±0.03; (n=15; ∗∗∗p<0.001) in N41 cells (Figure 5). In
untreated controls, this ratio remained constant at 0.70 ±0.01
in GT1-7 cells (n=10), 0.70 ±0.009 in N39 cells (n=10)
and 0.70 ±0.01 in N41 cells after the same period (n=15)
(Figure 5). It should be noted that the strongest increase of Ca2+
concentration was detected in the N41 cells (p≤0.0001) which
also has the highest expression level of adrenergic receptors and
TRPs.
In the next step, we evaluated 3-T1AM effects on whole-cell
currents of N41 cells to determine if increases in their magnitude
underlie rises in plasma membrane Ca2+influx in this cell
line. At−60 mV, 10 µM 3-T1AM increased inward currents from
−14.38 pA/pF to −60.78, which are attributable to Ca2+influx
because of the internal Ca2+free solution. At +130 mV, outward
rectifying currents strongly increased from 83.71 to 177.38 pA/pF
in the presence of 3-T1AM.
3-T1AM Mediates Rises in Ca2+Influx and
Whole-Cell Currents Through TRPM8
Activation
Previous studies demonstrated that 3-T1AM affects TRPM8
activation at a constant temperature in different cell types (21,
23). To validate that the Ca2+increase stems from an increase
in TRPM8 channel activity, N41 cells, which had the maximum
response to 3-T1AM stimulation, were exposed for 30 min to
10 µM BCTC, followed by bath supplementation with 10 µM
3-T1AM. Under these conditions, the TRPM8 channel blocker
abolished a 3-T1AM-induced Ca2+rise. More specifically, the
f340nm/f380nm ratio decreased from 1.81 ±0.04 to 0.93 ±0.02
in the presence of BCTC (n=15) (p≤0.001) (Figure 6A and
Supplemental Figure 5).
Our previous study demonstrated the inverse association
between TRPM8 and TRPV1 induced by 3-T1AM (21,22).
Different observations showed BCTC acts as a non-specific
TRPV1 inhibitor (45,46). Here, we also demonstrated the high
gene expression of TRPV1 in murine hypothalamic cell lines.
To rule out the involvement of TRPV1 in the 3-T1AM-induced
intracellular Ca2+response, we used AMTB as a high selective
TRPM8 blocker and capsazepine (CPZ) as a specific TRPV1
blocker. In the presence of 10 µM AMTB, the f340nm/f380nm ratio
decreased from 1.83 ±0.08 to 1.12 ±0.05 (Figure 6B), whereas
10 µM CPZ had no significant inhibitory effect on 3-T1AM-
induced intracellular Ca2+response (Supplemental Figure 6).
As AMTB suppressed a 3-T1AM-induced Ca2+increase,
we validated this effect by determining if this inhibitor
influenced underlying whole-cell currents. In the presence of
10 µM AMTB, inward currents decreased to −14.61 pA/pF
and outward currents decreased to 82.40 pA/pF (Figure 7).
Considering all these findings, 3-T1AM increased intracellular
Ca2+concentration and whole-cell currents in mouse
hypothalamic cells, thus confirming similar effects in other cell
types.
DISCUSSION
Administration of 3-T1AM in mice results in reversible effects
such as reduction of body temperature, cardiac output, and the
respiratory quotient along with anapyrexia and hyperglycemia
(1,47). There is evidence that 3-T1AM accumulates in the
Frontiers in Endocrinology | www.frontiersin.org 6September 2018 | Volume 9 | Article 523
Bräunig et al. 3-Iodothyronamine-Induced Hypothalamic Signaling Network
FIGURE 4 | Expression pattern of TRPM channels in (A) GT1-7, (B) N39 and (C) N41. Results of a SYBR Green based qPCR. Graphs show the ratio between the
reference gene Pgk1 and the TRPM channels and the Trpv1. The three hypothalamic cell lines show a resemblance of expression pattern. Trpm4 has always the
highest expression. A cluster of Trpm7,Trpm8, and Trpv1 display the second highest ratios compared to Pgk1.Trpm1,Trpm2, and Trpm3 also form a cluster with
similar expressions, however clearly lower expressed than Trpm7,Trpm8, and Trpv1.Trpm6 is detected at with the least mRNA content of all TRPMs. Trpm5 is not
expressed in these cell lines. Data was pooled from three independent experiments measured in duplicates.
FIGURE 5 | 3-T1AM induces Ca2+influx in GT1-7, N39 and N41 cell lines. Changes in cytosolic free Ca2+are depicted as the ratio of the fluorescence induced by
the excitation wavelength at 340 and 380 nm. 10 µM 3-T1AM induces an increase on intracellular Ca2+concentration in (A) GT1-7 (n=10), (B) N39 (n=15) and
(C) N41 (n=15) cells. Notably, 10 µM 3-T1AM induces a significantly larger increase in intracellular Ca2+in N41 cell line. Without compound application, no changes
in Ca2+influx could be observed (n=10). Compounds were added to cells at the time points indicated by the arrow and nindicates number of the single cells.
Experiments were performed in 400s. The total number of cells was collected in five independent experiments. Values represent mean ±SEM.
hypothalamic nuclei (6,7). The aim of this study was to
explore the underlying mechanism behind the stimulatory effect
of this thyroid hormone metabolite in selected hypothalamic
regions.
3-T1AM-Induced Signalosome Activates
PVN Neurons of C57BL/6 Mice
Within the hypothalamus, PVN is one of the most extensively
studied nuclei and is playing a pivotal role in the control of
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Bräunig et al. 3-Iodothyronamine-Induced Hypothalamic Signaling Network
FIGURE 6 | TRPM8 mediates 3-T1AM-induced Ca2+response in N41 cell line. Cells were pre-incubated with inhibitors (10 µM AMTB or 10 µM BCTC) 30 min before
the measurement. Stimulation was performed with 10 µM 3-T1AM and Ca2+influxes were measured (n=15–19) with and without the inhibitors. (A) 3-T1AM
increased Ca2+influx and pre-incubation with BCTC significantly suppressed this effect. (B) AMTB showed the similar inhibitory effect on 3-T1AM-induced Ca2+
influx. Experiments were performed in 400s. Compounds were added to cells at the time points indicated by the arrow and n indicates number of the single cells. The
total number of cells was collected in 5 independent experiments. Values represent mean ±SEM.
FIGURE 7 | 3-T1AM activates whole-cell currents in N41 cell line. (A) Time course of whole-cell currents at −60 mV (lower trace) and 130 mV (upper trace) showing
the current activation by 10 µM 3-T1AM (left). (B) Original traces of 3-T1AM activated current responses to voltage ramps from −60 mV up to +130 mV (right).
Currents are shown before application (labeled as 1) and during application of 1 µM 3-T1AM (labeled as 2) and in the presence of 10 µM AMTB (labeled as 3). (C)
10 µM 3-T1AM increased inward and outward whole-cell channel currents in N41 cells and this effect was strongly suppressed in the presence of 10 µM AMTB.
Currents are shown before application (control), during application of 10 µM 3-T1AM and in the presence of 10 µM AMTB (n=10). Whole-cell currents were recorded
using step and ramp protocols involving voltage steps of 10 mV ranging between−60 to +130 mV for 400 ms. The currents were normalized to capacitance to obtain
current density (pA/pF). Statistical significance was determined by one-way ANOVA, comparing the basal current density (pA/pF) against 3-T1AM and AMTB. Data are
the mean ±SEM of at least 10 independent experiments; *p≤0.05.
fluid homeostasis, lactation, cardiovascular regulation, feeding
behavior, nociception and response to stress (11). It has already
been shown that the direct injection of 3-T1AM into the
lateral ventricle of male mice leads to the activation of neurons
in the anterior commissural nucleus of hypothalamus (48).
Here, we observed that intraperitoneal injection of 3-T1AM
results in the activation of PVN neurons in C57BL/6 mice
(Figure 1). Recently, Gachkar et al. showed that 3-T1AM-
induced hypothermia is due to vasodilation, which is not
directly induced in the veins. In this study they suggested
that 3-T1AM might induce the tail vasodilation through
central action in male mice (14). Here, we investigated the
loci in the brain, which are known to be involved in the
regulation of the body temperature through non-shivering
and shivering thermogenesis (PAG and DMH) and heat
dissipation like vasodilation [VTA, (49)]. The result of this study
demonstrated no differences in c-FOS activation in these areas
(Figure 1B).
Previous studies detected no effect of 3-T1AM metabolites
such as 3-iodothyroacetic acid and iodine-free thyronamine
on cardiac output or thermoregulation (50,51). Nonetheless,
whether 3-T1AM directly activates the PVN neurons or causes
the activation of neuronal projections from other nuclei is
uncertain. Moreover, the exact cellular mechanism initiated by
3-T1AM once it reaches in the hypothalamic nuclei is yet to be
discovered.
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Bräunig et al. 3-Iodothyronamine-Induced Hypothalamic Signaling Network
3-T1AM Slightly Stimulates GαsSignaling
in Murine Hypothalamic Cell Lines
Aminergic receptors are largely expressed in the hypothalamus
and play a substantial role in various regulatory responses,
such as metabolic regulations (52). These receptors are
known 3-T1AM targets and previous studies assumed that
pharmacological effects of this thyroid hormone metabolite are
attributable to aminergic receptor signaling (1,16–19). It has
been reported that 3-T1AM enhances Gαssignaling in response
to ISOP co-stimulation at ADRB2 (17) and activates Gi/o
signaling combined with NorEpi of the ADRA2A in transfected
HEK293 cells (16). Here, we could not detect the activation of
Gi/oin response to the 3-T1AM in murine hypothalamic cell
lines (Figure 3B). Nevertheless, N39 and N41 showed an FSK-
amplified Gαssignal due to 3-T1AM stimulation (Figure 3B).
The low GPCR expression rate could be the reason for the lack
of strong Gαsin these cells (Figure 2). The absence of Gi/osignal
through 5-HT1b could be explained by dimerization with TAAR1
(19). It was shown that 3-T1AM is capable of inducing a clear Gi/o
signal in HEK293 cells overexpressing 5-HT1b. However, co-
expression of 5-HT1b with TAAR1 abrogated 3-T1AM-induced
cAMP signaling (19). Since the three hypothalamic cell lines all
co-express TAAR1 and 5-HT1b, the Gi/osignal of 5-HT1b via
3-T1AM stimulation is probably disrupted. The FSK stimulated
N39 and N41 cells showed a significant increase in cAMP
concentration after 3-T1AM challenge (Figure 3B). Besides
its own adenylyl cyclase activating property, FSK stimulation
additionally favors activation of adenylyl cyclase through Gαs
(44,53,54). Here, we concluded that 3-T1AM induces a weak
Gαssignal in N39 and N41 cells.
3-T1AM Activates TRPM8 Channel in
Murine Hypothalamic Cell Lines
Ca2+mobilization in different cell types plays an essential role in
the modulation of c-FOS gene expression (55,56). In neurons,
Ca2+influx is a prerequisite for activation of the ERK/MAPK
pathway. It is known that c-FOS activation reflects summation
or integration of this Ca2+dependent-neuronal activity (57,58).
Moreover, Ca2+acts synergistically with cAMP to activate c-FOS
transcription (59). To test whether 3-T1AM induces an increase
of intracellular Ca2+in hypothalamic cell lines, we evaluated ion
channel activation induced by 3-T1AM.
It is known that GPCR activation induces an increase of
intracellular Ca2+concentration through different pathways
(60–63). Recent studies demonstrated the co-expression of
GPCRs and TRPs in a variety of cell types (31). Different
signaling intermediates such as adaptor proteins, kinases, and
lipid metabolites functionally link GPCRs to TRPs (30,64).
Various TRPs play a pivotal role in the mechanisms that are
involved in energy metabolism and temperature adaptation (26,
65,66). Some recent studies reported that activation of warm-
sensitive TRPM2 leads to a similar thermoregulatory response
as the one observed in mice after systemic administration
of 3-T1AM (67). Previously, we showed that 3-T1AM acts
as a cooling agent to directly affect TRPM8 activation in
different cell types (21,22). In rat thyrocyte, 3-T1AM induced
Ca2+responses similar to specific TRPM8 agonists such as
menthol (68). In ocular cells, 3-T1AM evoked Ca2+mobilization
and increases in whole-cell currents, a stimulatory effect that
could be specifically attenuated in the presence of specific
TRPM8 blocker (21,22). The result of this study showed
that 3-T1AM induces intracellular Ca2+increase through the
TRPM8 channel in murine hypothalamic cell lines. Finding
the functional link between 3-T1AM and this specific TRP
channel in hypothalamic cell lines is relevant since there are
indications that the TRPM8 channel regulates energy metabolism
in different tissues and plays a crucial role in thermoregulation
(69,70). TRPM8 stimulation, for instance, induces mitochondrial
activation and heat production in brown adipocytes. Chronic
TRPM8 agonist administration enhances the energy metabolism
in brown adipocytes and prevents obesity in mice (69). In
skeletal muscles, TRPM8 activation by dietary menthol improves
energy metabolism through Ca2+-dependent upregulation of
the peroxisome proliferator-activated receptor-γcoactivator 1α
(PGC1α) which is involved in the mitochondrial function
(70). Recently, it has been demonstrated that TRPM8-deficient
mice develop late-onset obesity and metabolic dysfunction at
moderate cooling, suggesting the importance of TRPM8 in the
coupling between thermoregulation and energy homeostasis.
Nevertheless, Trpm8−/−mice exhibit a remarkable decrease
of core body temperature due to increased tail heat loss (71)
which is in contrast to the 3-T1AM stimulatory effect on
TRPM8 found in our in vitro electrophysiological observations.
Therefore, we investigated the possible effect of 3-T1AM on
TRPV1 as another known thermo-sensitive TRP channel as it
has been shown that there is an interplay between TRPV1 and
TRPM8 (72).
Previous studies demonstrated the co-expression of TRPV1
with TRPM8 in different cell types including rat hippocampal
neurons, intralobar pulmonary arteries, aorta, neuroendocrine
tumor cells, retinoblastoma cells, uveal melanoma cells, and
corneal cells (36,73–76). Here, we showed the co-expression of
these two thermo-TRP channels in three different hypothalamic
cell lines. It is well established that there is a cross talk
between TRPM8 and TRPV1 channels in various tissues. For
instance, TRPM8 agonist blocks the mechanical and heat
hyperalgesia caused by TRPV1 activation (77,78). It was also
demonstrated that icilin, a specific TRPM8 agonist, attenuates
TRPV1-dependent calcitonin gene-related peptide release in the
colon and is suggested as a promising therapeutic target for the
treatment of colitis (79). The interdependence of the TRPM8 and
TRPV1 ion channel function as well as the role of both channels
in thermo-regulation have raised the question as to which TRP
channel is the main target of 3-T1AM in hypothalamic cell lines.
The results of this study clearly showed that the effect of 3-T1AM
were not attributable to TRPV1 since only the specific TRPM8
blocker (AMTB) could strongly inhibit the 3-T1AM-induced
Ca2+influx and whole-cell current.
Collectively, the results of this study demonstrated the
Ca2+signal transduction pathways induced by 3-T1AM and
provided evidence of TRP channel modulation via this TH
derivative in hypothalamic cell lines. Characterization of the
intracellular signaling cascade induced by 3-T1AM might explain
Frontiers in Endocrinology | www.frontiersin.org 9September 2018 | Volume 9 | Article 523
Bräunig et al. 3-Iodothyronamine-Induced Hypothalamic Signaling Network
the underlying mechanism behind the profound physiological
effects of this metabolite.
AUTHOR CONTRIBUTIONS
HB, NK, and JB designed the study. JB, SJ, and NK performed the
experiments and analyzed the data. SJ, CH, and JM performed
the mouse studies. NK, JB, SM, and HB wrote and edited the
manuscript. CH, JM, and MR discussed data and edited the
manuscript. All authors approved the manuscript.
FUNDING
This work was supported by the Deutsche
Forschungsgemeinschaft (DFG), the priority program SPP1629
Thyroid Trans Act BI893/5-2 and ME1706/13-1. SM was
also supported by DFG about a project related to TRPs
(ME1706/18-1).
ACKNOWLEDGMENTS
The authors would like to thank Cigdem Cetindag (Charité,
Institute of Experimental Pediatric Endocrinology) for the
technical assistance.
SUPPLEMENTARY MATERIAL
The Supplementary Material for this article can be found
online at: https://www.frontiersin.org/articles/10.3389/fendo.
2018.00523/full#supplementary-material
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Conflict of Interest Statement: The authors declare that the research was
conducted in the absence of any commercial or financial relationships that could
be construed as a potential conflict of interest.
Copyright © 2018 Bräunig, Mergler, Jyrch, Hoefig, Rosowski, Mittag, Biebermann
and Khajavi. This is an open-access article distributed under the terms of the Creative
Commons Attribution License (CC BY). The use, distribution or reproduction in
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are credited and that the original publication in this journal is cited, in accordance
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