EBioMedicine 8 (2016) 60–71

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EBioMedicine
journal homepage: www.ebiomedicine.com

Research Paper

Catecholamines Facilitate Fuel Expenditure and Protect Against Obesity
via a Novel Network of the Gut-Brain Axis in Transcription Factor
Skn-1-deﬁcient Mice
Shota Ushiama a,1, Yoshiro Ishimaru a,⁎,1, Masataka Narukawa a,1, Misako Yoshioka a, Chisayo Kozuka b,
Naoki Watanabe c, Makoto Tsunoda d, Naomi Osakabe c, Tomiko Asakura a, Hiroaki Masuzaki b, Keiko Abe a,e,⁎a

Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan
Division of Endocrinology, Diabetes and Metabolism, Hematology, Rheumatology (Second Department of Internal Medicine), Graduate School of Medicine, University of the Ryukyus, 207 Uehara,
Nishihara, Okinawa 903-0215, Japanc
Department of Bio-science and Engineering, Shibaura Institute of Technology, 307 Fukasaku, Minuma-ku, Saitama 337-8570, Japand
Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japane
Kanagawa Academy of Science and Technology, Takatsu-ku, Kawasaki-shi, Kanagawa, Japanba r t i c l e

i n f oArticle history:
Received 25 January 2016
Received in revised form 23 April 2016
Accepted 25 April 2016
Available online 30 April 2016
Keywords:
Energy metabolism
Brush cells
Catecholamine
Insulina b s t r a c t
Taste signals and nutrient stimuli sensed by the gastrointestinal tract are transmitted to the brain to regulate feeding
behavior and energy homeostasis. This system is referred to as the gut-brain axis. Here we show that both brushcells and type II taste cells are eliminated in the gastrointestinal tract of transcription factor Skn-1 knockout (KO)mice. Despite unaltered food intake, Skn-1 KO mice have reduced body weight with lower body fat due to increased
energy expenditure. In this model, 24-h urinary excretion of catecholamines was signiﬁcantly elevated, accompanied
by increased fatty acid β-oxidation and fuel dissipation in skeletal muscle and impaired insulin secretion driven by glucose. These results suggest the existence of brain-mediated energy homeostatic pathways originating from brush cells
and type II taste cells in the gastrointestinal tract and ending in peripheral tissues, including the adrenal glands. The discovery of food-derived factors that regulate these cells may open new avenues the treatment of obesity and diabetes.
Research Context: Taste signals and nutrient stimuli sensed by the gastrointestinal tract are transmitted to the brain to
regulate feeding behavior and energy homeostasis along the gut-brain axis. We propose the concept that taste-receiving cells in the oral cavity and/or food-borne chemicals-receiving brush cells in the gut are involved in regulation of the
body weight and adiposity via the brain. The discovery of food-derived factors that regulate these cells may open new
avenues for the treatment of obesity and diabetes.
© 2016 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license(http://creativecommons.org/licenses/by-nc-nd/4.0/).

1. Introduction

Abbreviations: BAT, brown adipose tissue; ChgA, chromogranin A; CT, computed
tomography; Dbh, dopamine-β-hydroxylase; Dclk1, doublecortin-like kinase 1; Ddc,
dopa decarboxylase; GI, gastrointestinal; GIP, glucose-dependent insulinotropic peptide;
GLP-1, glucagon-like peptide-1; GSIS, glucose-stimulated insulin secretion; HFD, high-fat
diet; IPGTT, intraperitoneal glucose tolerance test; ITT, insulin tolerance test; KO,knockout; NEFA, non-esteriﬁed fatty acid; OGTT, oral glucose tolerance test; Pnmt,
phenylethanolamine N-methyltransferase; RER, respiratory exchange ratio; SCC, solitarychemosensory cells; T3, triiodothyronine; T4, tetraiodothyronine; TG, triacylglycerol; Th,tyrosine hydroxylase; Trpm5, transient receptor potential melastatin 5; TSH, thyroid
stimulating hormone; Ucp3, uncoupling proteins 3; WAT, white adipose tissue.
⁎ Corresponding authors at: Department of Applied Biological Chemistry, Graduate
School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyoku, Tokyo 113-8657, Japan.
E-mail addresses: ayishi@mail.ecc.u-tokyo.ac.jp (Y. Ishimaru),
aka7308@mail.ecc.u-tokyo.ac.jp (K. Abe).
1
These authors contributed equally to this work.

Taste signals and nutrient stimuli sensed by the gastrointestinal (GI)tract are transmitted to the central nervous system, including the nucleus of the solitary tract and hypothalamus, via afferent neurons and humoral mediators, thereby controlling feeding behavior and energy
homeostasis. This system is referred to as the gut-brain axis
(Cummings and Overduin, 2007; Furness, 2012). This information issubsequently conveyed from the brain to peripheral tissues via efferent
sympathetic and parasympathetic nerves including preganglionic sympathetic splanchnic input to the adrenal glands. For example, gut
incretins such as glucose-dependent insulinotropic peptide (GIP) andglucagon-like peptide-1 (GLP-1), which are released from the intestinal
enteroendocrine cells in response to nutrient ingestion, potentiate glucose-stimulated insulin secretion (GSIS) from pancreatic β-cells (Wuet al., 2015). GLP-1 also activates the GLP-1 receptors expressed on
vagal afferent nerve terminals in the portal vein and in the brain across

http://dx.doi.org/10.1016/j.ebiom.2016.04.031
2352-3964/© 2016 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

S. Ushiama et al. / EBioMedicine 8 (2016) 60–71

the blood-brain barrier, which is involved in the regulation of appetite(Katsurada et al., 2014; Kinzig et al., 2002; Vahl et al., 2007). Gavage ofagonists for bitter taste receptor increases food intake via the secretionof ghrelin, a hormone that potentiates hunger sensation (Janssen et al.,2011).
The Skn-1 (also known as Pou2f3) gene, which encodes the POU
homeodomain transcription factor, was originally identiﬁed as a regulator of the differentiation of epidermal keratinocytes (Andersen et al.,
1993, 1997). We previously reported that Skn-1a is expressed insweet, umami (savory), and bitter-sensing taste cells, which are referred to as type II taste cells (Matsumoto et al., 2011). Type II taste
cells are completely eliminated in Skn-1 knockout (KO) mice, resulting
in loss of electrophysiological and behavioral responses to sweet,
umami, and bitter tastes. Thus, Skn-1a is critical for generating type II
taste cells. Skn-1a is also required for the generation of Trpm5-expressing solitary chemosensory cells (SCCs) in the nasal respiratory epithelium and microvillous cells in the main olfactory epithelium (Ohmoto etal., 2013; Yamaguchi et al., 2014). Skn-1a is expressed in several tissues
including the stomach, but not in the brain (Yukawa et al., 1993).
There are four major cell types in the small intestine: enterocytes
and Goblet, Paneth, and enteroendocrine cells (van der Flier andClevers, 2009). In addition to these cell types, brush cells (also referred
to as tuft cells or caveolated cells) constitute a minor fraction (0.4%) of
the adult mouse intestinal epithelium (Gerbe et al., 2012). Brush cells
are supposed to be chemosensory cells (Young, 2011) and express transient receptor potential melastatin 5 (Trpm5), Doublecortin-like kinase
1 (Dclk1), and choline acetyltransferase (Bezencon et al., 2008; Gerbe
et al., 2009, 2011; Saqui-Salces et al., 2011; Schütz et al., 2015). The
mechanisms of differentiation and the function of brush cells remain
elusive.
In the present study, we found that Skn-1a regulates differentiationof Trpm5-expressing brush cells in the GI tract. Skn-1 KO mice exhibited
reduced body weight with lower body fat than wild-type (WT) littermates. Despite unaltered food intake, Skn-1 KO mice exhibited increased energy expenditure, caused by augmented catecholamine
secretion. Our work raises the concept that taste cells receiving sweet,
bitter, and umami tastes as well as brush cells receiving food-borne
chemicals are involved in regulating body weight and body fat. Collectively, the present study provides new insights into the regulation of energy homeostasis originating from brush cells and taste cells in the GI
tract and signaling to peripheral tissues including the adrenal glands,
via the brain.
2. Material and Methods
2.1. Experimental Animals
All animal experiments were approved by the Animal Care and Use
Committee at The University of Tokyo. Skn-1/Pou2f3-deﬁcient micewith a mixed 129 × C57BL/6J background were generated as previously
described (Matsumoto et al., 2011) and then backcrossed to C57BL/6J
for N 10 generations. Male Skn-1 KO mice and their WT littermates generated by crossing the heterozygous mice were used in the present
study. Mice were fed a normal chow diet (Lab MR Breeder, Nosan Co.,
Yokohama, Japan) after weaning or High-Fat Diet 32 (CREA Japan Inc.,Tokyo, Japan) from 4 weeks of age for N12 weeks to induce dietary obesity. Mice were maintained in a room with a constant temperature of
22 ± 1 °C under a 12-h light-dark cycle (lights on at 8 a.m.). Tissue samples were collected from 12- to 16-week-old mice that were fasted for
18 h.
2.2. X-ray Computed Tomography (CT) Scan Analysis
Mice older than 16 weeks were anesthetized using an isoﬂurane
nebulizer (Muromachi Kikai Co. Ltd., Tokyo, Japan); a Latheta LCT-200

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CT instrument (Aloka-Hitachi LCT-200, Tokyo, Japan) was then used toscan abdominal fat and muscle mass and extract thighbone density.
2.3. Blood Chemical Parameters
Blood was collected from approximately 20-week-old mice by cardiac puncture at 10 a.m. under ad libitum feeding condition and after 18-h
fasting. Serum chemical parameters and hormones (PTH, T3, and T4)
were analyzed by Nagahama Life Science Laboratory (Shiga, Japan).
Plasma insulin and leptin were measured using the Luminex 200™ System (Luminex, Austin, TX, USA) by GeneticLab Co., Ltd. (Sapporo,
Japan). Plasma adiponectin and FGF21 were measured using mouseadiponectin ELISA (BioVender, Brno, Czech Republic) and mouse andrat FGF-21 ELISA (BioVender), respectively.
2.4. Indirect Calorimetry
Indirect calorimetry was performed on 20- to 28-week-old miceusing an indirect calorimeter (ARCO-2000, Arco Systems Inc., Chiba,
Japan) and spontaneous motor activity was measured using a pyroelectric infrared ray sensor (NS-AS01, Neuroscience Inc., Tokyo, Japan) for
3–4 days after 2 days of habituation essentially as described by Tschop
et al. (2011) or with an indirect calorimeter (MK-5000RQ, Muromachi
Kikai Co. Ltd.) as previously described (Watanabe et al., 2014). The respiratory exchange ratio (RER) was calculated as the molar ratio of
VCO2/VO2. Energy expenditure (kcal per hour) was calculated as heat(kcal/h) = (3.815 + 1.232 × RER) × VO2.
2.5. Mitochondrial DNA Copy Number
To measure the mitochondrial DNA copy number, the ratio of mitochondrial DNA to genomic DNA was calculated by a previously described method (Watanabe et al., 2014). In brief, total DNA wasextracted from the gastrocnemius of 14- to 19-week-old mice thatwere fasted for 16 h using a QIAamp DNA mini kit (QIAGEN Ltd.,Tokyo, Japan) and used for qPCR (Applied Biosystems Japan Ltd.,Tokyo, Japan). We used the TaqMan Gene Expression Assay (Applied
Biosystems Foster City, USA; ACTB, Mm00607939_s1; CYTB,
Mm044225274). The PCR cycling conditions were 50 °C for 2 min, 95 °C for 10 min, and 40 cycles of 95 °C for 15 s and 60 °C for 1 min. The mitochondrial DNA copy number is presented relative to nuclear DNA following ampliﬁcation of the mitochondrial gene region (cytochrome b
vs. the nuclear endogenous control region, β-actin).
2.6. DNA Microarray
DNA microarray analysis using total RNA extracted from the gastrocnemius of 12- to 16-week-old mice that were fasted for 18 h was performed as previously described (Nakai et al., 2008) with minor
modiﬁcations. In brief, Total RNA samples (100 ng each) were preparedand processed for microarray analysis using a 3′ IVT Express kit and
GeneChip® Mouse Genome 430 2.0 Array (Affymetrix, Santa Clara, CA,
USA) according to the manufacturer's standard protocols. The raw microarray data (CEL ﬁles) were quantiﬁed by the Factor Analysis for Robust Microarray Summarization (FARMS) (Hochreiter et al., 2006)
using the statistical language R (Team and Computing, 2005) andBioconductor (Gentleman et al., 2004). To identify differentiallyexpressed genes, the rank products (RP) method (Breitling et al.,2004) was applied to the FARMS-quantiﬁed data. All microarray data
are MIAME compliant and have been deposited in a MIAME-compliant
database, the National Center for Biotechnology Information (NCBI)
Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo/, GEO Series accession number GSE76936), as described in more detail on the
MGED Society web site (http://www.mged.org/Workgroups/MIAME/
miame.html).

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2.7. Fecal TG and Total Energy Content
Fecal lipids were extracted from dried feces of 15-week-old mice fed
a HFD according to the Folch method (Folch et al., 1957), and the TG
concentration was measured using the Wako triglyceride E-Test(Wako Pure Chemical Industries, Ltd., Osaka, Japan). The total energy
content of feces collected for 8 weeks was measured by bomb
calorimetry.

catecholamine, 60 μL of hydrochloric acid (6 mol/L) was added to thecollection vials (Moreira-Rodrigues et al., 2012). The collected urine
was puriﬁed using MonoSpin PBA (GL Sciences, Tokyo, Japan) andurine catecholamines were measured by HPLC as previously described(Tsunoda et al., 2011). In brief, Chromatography was performed using
an Inertsil ODS-4 column (5 μm, 250 × 20 mm i.d., GL Science) with acetate-citrate buffer at a ﬂow rate of 0.5 mL/min and detected by electrochemical detection. The concentration of urine creatinine was measured
using a LabAssay™ Creatinine kit (Wako Pure Chemical Industries, Ltd.).

2.8. Measurement of Urine Catecholamines
2.9. qRT-PCR in Adrenal Glands
Twenty-four-hour urine (for catecholamine measurements) wascollected into collection vials from 11- to 12-week-old mice individuallyhoused in metabolic cages. To avoid spontaneous oxidation of

Total RNA was extracted from adrenal glands using RNeasy mini-columns (Qiagen, Venlo, Netherlands), followed by genomic DNA

Fig. 1. Brush cells are eliminated in the GI tract of Skn-1 KO mice. (a) Tissue distribution of Skn-1a. RT-PCR analysis revealed that Skn-1a was abundantly expressed in the circumvallate
papillae, stomach, duodenum, jejunum, and ileum but absent or only faintly expressed in all other tissues examined, including the pancreas, islets of Langerhans, brain, liver, skeletal
muscle, white adipose tissue (WAT), and brown adipose tissue (BAT). (b) Immunostaining revealed strong Skn-1a signals in the corpus beneath the limiting ridge in the stomach(upper panel) and in a subset of cells in the small intestine (lower panel). Inset in the upper right corner of each panel shows higher magniﬁcation image of the area enclosed by thedashed box. (c) In situ hybridization revealed that Trpm5 mRNA was expressed in the jejunum of WT mice (upper panel) but not in Skn-1 KO mice (lower panel). (d) Immunostaining
revealed Dclk1 signals in WT mice (upper panel) but not Skn-1 KO mice (lower panel). The arrowheads indicate signals. (e) Double immunostaining demonstrated that nearly all
Trpm5-positive cells were also positive for Skn-1a. The arrows indicate cells expressing Trpm5 and Skn-1a. Scale bars: 100 μm and 25 μm (inset). See also Fig. S1.

S. Ushiama et al. / EBioMedicine 8 (2016) 60–71

digestion using RNase-free DNase (Qiagen). First-strand cDNA was generated by reverse transcription from total RNA (Superscript III Reverse
Transcription Kit; Life Technologies, Gaithersburg, MD). Levels ofmRNA transcripts were determined by qPCR (ABI Prism 7000 Sequence Detection System, Applied Biosystems, Foster City, CA).
cDNA levels were normalized against β-actin. PCR ampliﬁcation
was performed using TaqMan technology (TaqMan Gene Expression

63

Assays, Applied Biosystems). The TaqMan probe IDs were as
follows: tyrosine hydroxylase (Th; Mm00447557_m1), dopa
decarboxylase (Ddc; Mm00516688_m1), dopamine β-hydroxylase(Dbh; Mm00460472_m1), phenylethanolamine N-methyltransferase (Pnmt; Mm00476993_m1), and β-actin (Mm00607939_s1).
The delta-delta method was used for relative quantiﬁcation (Livakand Schmittgen, 2001).

Fig. 2. Metabolic phenotypes of Skn-1 KO mice fed a normal chow diet. (a) Changes in body weight under normal chow diet conditions. When fed a normal chow diet, Skn-1 KO mice had
lower body weight than their WT littermates for 13 weeks after weaning (n N 75). Also see Fig. S2A. (b) Skn-1 KO mice had a lower body fat percentage than their WT littermates, whereas
there was no signiﬁcant difference in muscle mass percentage between the two genotypes (n = 10–19). (c and d) Serum levels of total ketone bodies were higher in Skn-1 KO mice thantheir WT littermates under ad libitum feeding (n = 18–28) (c) and 18-h fasting conditions (n = 13) (d). (e) Skn-1 KO and WT littermates had similar food intake at 8 weeks of age (n = 5–
8). (f) Skn-1 KO mice exhibited increased energy expenditure compared with their WT littermates (n = 9). (g) qRT-PCR analysis revealed that the mitochondrial DNA content in skeletal
muscle was signiﬁcantly higher in Skn-1 KO mice than in their WT littermates. The data are presented as the mean ± SEM; **p b 0.01 and *p b 0.05. See also Fig. S2.

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2.10. OGTT, IPGTT, ITT, and Measurement of Insulin, GIP, and GLP-1
Gavage administration of glucose (3.0 mg/g of body weight for measurement of blood glucose, plasma insulin, and plasma total GIP;
5.0 mg/g of body weight for measurement of plasma active GLP-1;
2.0 mg/g of body weight for measurement of blood glucose and plasma
insulin in mice fed a HFD) was performed on 16-week-old WT and Skn1 KO mice at 10 a.m. after 18-h fasting (n = 20–30 animals per group)
using a syringe attached to a feeding needle (KN-349-M1, Natsume
Seisakusho Co. Ltd., Tokyo, Japan) inserted through the mouth into thestomach. For IPGTT or ITT, mice were fasted for 18 or 4 h, then injected
intraperitoneally with glucose (1.5 mg/g of body weight) or insulin(0.75 mU/g of body weight), respectively. Tail blood samples were collected from the tail vein before gavage (time 0) and 15, 30, 45, 60, 90,
and 120 min after gavage. For measurement of GIP and GLP-1, tailblood samples were collected into chilled tubes containing DPP-IV Inhibitor (Merck Millipore, Billerica, MA) and/or EDTA and aprotinin(for GLP-1; Wako Pure Chemical Industries, Ltd.) before gavage (time0) and 15, 30, and 60 min after gavage (for GIP) or 10, 20, and 40 min
after gavage (for GLP-1). Blood glucose, plasma insulin, plasma total
GIP, and plasma active GLP-1 were measured by Glucose Pilot (Iwai
Chemicals Co. Ltd., Tokyo, Japan), Ultra Sensitive Mouse Insulin ELISA
Kit (Morinaga Institute of Biological Science Inc., Kanagawa, Japan),
Rat/Mouse GIP (Total) ELISA Kit (EZRMGIP-55K, Merck Millipore), and
GLP-1 (Active) ELISA Kit (AKMGP-011, Shibayagi Co. Ltd., Gunma,
Japan), respectively.
2.11. RT-PCR
First-strand cDNAs from 11 different tissues except the circumvallate papillae (Genostaff, Tokyo, Japan) were used for RT-PCR. For the circumvallate papillae, total RNA was extracted and reverse-transcribed
into cDNA with an oligo dT primer. Approximately 500 bp of coding region encompassing multiple exons of Skn-1a was ampliﬁed from each
cDNA for 30 cycles at an annealing temperature of 60 °C. The volumeof PCR products for Skn-1a loaded on the agarose gel was approximately
normalized using the PCR product for GAPDH.

of the antibody (data not shown). The other primary antibodies usedwere as follows: rabbit anti-Skn-1a antibody (#sc-330, Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-Dclk1 antibody (#AP7219b,
Abgent, San Diego, CA), rabbit anti-ChgA antibody (#20085,
ImmunoStar, Hudson, WI), and goat anti-GLP-1 antibody (#sc-7782,
Santa Cruz Biotechnology). Immunohistochemistry was performed aspreviously described (Ishimaru et al., 2006). In brief, fresh-frozen sections (10-μm thick) were ﬁxed in 4% paraformaldehyde (PFA), permeabilized with ice-cold methanol, blocked with phosphate-bufferedsaline (PBS) containing 5% skim milk, and incubated with rabbit antiSkn-1a antibody (1/1000 dilution), rabbit anti-Trpm5 antibody (1/200
dilution), rabbit anti-Dclk1 antibody (1/50 dilution), or rabbit antiChgA antibody (1/200 dilution), followed by incubation with Alexa
Table 1
Up-regulated genes related to mitochondrial function in skeletal muscle.
Gene symbol

ATP-binding cassette, sub-family D(ALD), member 2
Ociad2OCIA domain containing 2
Pptc7
PTC7 protein phosphatase homolog (S.
cerevisiae)
4930402E16Rik RIKEN cDNA4930402E16Rik gene8430408G22Rik RIKEN cDNA 8430408G22 gene
Tbc1d15
TBC1 domain family, member 15
Xaf1
XIAP associated factor 1
Col4a3bp
Collagen, type IV, alpha 3(Goodpasture antigen) binding
protein
Dsp
Desmoplakin
DlaT

Fdx1
Gpam
Hk2
Coq10bImmt

2.12. Islet Isolation and In Vitro Insulin Release From Mouse Islets
Isolated pancreatic islets were prepared by liberase (Roche Diagnostics, Basel, Switzerland) digestion, manually selected, and incubated in
RPMI 1640 tissue culture medium (Life Technologies) supplemented
with 10% (vol/vol) fetal bovine serum at 37 °C overnight. Insulin secretion in vitro was measured in static incubations. Prior to experiments,
islets were pre-incubated for 60 min at 37 °C in Krebs-Ringer (KR) buffer composed of 119 mM NaCl, 4.7 mM KCl, 2.5 mM CaCl2, 1.2 mM MgCl2,
1.2 mM KH2PO4, 25 mM NaHCO3, and 10 mM HEPES (pH 7.4) with 5%
bovine serum albumin. The medium was gassed with 95% O2 and 5%
CO2 to obtain constant pH and oxygenation. The groups of 10 islets
were incubated for 60 min at 37 °C in Krebs-Ringer buffer solution containing 2.5 mM glucose (low glucose). The islets were transferred to
new low glucose Krebs-Ringer buffer and incubated for 60 min. Subsequently, the islets were incubated in Krebs-Ringer buffer containing
25 mM glucose (high glucose) for 30 min. The supernatants from thesecond low-glucose solution and from the high-glucose solution werecollected for the insulin ELISA assay and stored at −80 °C. Insulin concentrations were measured using an Ultra Sensitive Mouse Insulin
ELISA Kit (Morinaga Institute of Biological Science).
2.13. Immunohistochemistry
Rabbits were immunized with peptides corresponding to residues
1140–1153 (C+PASARDREYLESGL) of Trpm5 (Operon Biotechnologies,
Tokyo, Japan). Preincubation of the antibody with peptide antigen(10 ng/mL) abolished the brush cell staining, conﬁrming the speciﬁcity

Gene name

ABCD2

Ide
Krt5
Kif1b

Elovl6
Csde1
Prkca

Ppm1k
Ppp3ca
Reep1
ND5
Opa1
Slc25a12

Slc25a25

Tmtc1
Tap1
Ucp3

Dihydrolipoamide S-acetyltransferase(E2 component of pyruvate
dehydrogenase complex)
Ferredoxin 1
Glycerol-3-phosphate acyltransferase,mitochondrial
Hexokinase 2
Coenzyme Q10 homolog B (S.
cerevisiae)
Inner membrane protein,
mitochondrial
Insulin degrading enzyme

Probe ID
1419748_at
1435917_at
1455958_s_at
1459869_x_at
1433837_at
1416062_at
1443698_at
1420384_at

1435493_at,
1435494_s_at
1426264_at,
1426265_x_at
1449108_at
1419499_at
1422612_at
1460510_a_at
1429533_at

1423121_at,
1435140_at
Keratin 5
1424096_at
Kinesin family member 1B
1423995_at,
1451642_at,
1451200_at,
1425270_at
ELOVL family member 6, elongation of 1417403_atlong chain fatty acids (yeast)
Cold shock domain containing E1, RNA 1423997_at
binding
Protein kinase C, alpha1437393_at,
1446598_at,
1450945_atProtein phosphatase 1K (PP2C domain 1441988_at
containing)
Protein phosphatase 3, catalytic
1426401_at,
subunit, alpha isoform
1438478_a_at
Receptor accessory protein 1
1433509_s_at
NADH dehydrogenase subunit 51426088_at
Optic atrophy 1
1434890_at
1436440_at
Solute carrier family 25(mitochondrial carrier, Aralar),member 12
1424735_at,
Solute carrier family 25
1447856_x_at(mitochondrial carrier, phosphate
carrier), member 25
Transmembrane and tetratricopeptide 1435261_at
repeat containing 1
Transporter 1, ATP-binding cassette,
1416016_at
sub-family B (MDR/TAP)
Uncoupling protein 3 (mitochondrial, 1420658_at
proton carrier)S. Ushiama et al. / EBioMedicine 8 (2016) 60–71

Fluor 488- (#A21206) or Alexa Fluor 555-conjugated anti-rabbit IgG(#A31572, Life Technologies). For GLP-1, fresh-frozen sections (10-μm
thick) were ﬁxed with 4% PFA, blocked with PBS containing 5% normal
donkey serum and 0.2% Triton X-100, and incubated with goat antiGLP-1 antibody (1/150 dilution) followed by incubation with Alexa
Fluor 555-conjugated anti-goat IgG (#A21432, Life Technologies).
For double staining, fresh-frozen sections (10-μm thick) were ﬁxed
with 4% PFA and permeabilized with ice-cold methanol followed by antigen retrieval. The sections were then blocked with Tris-buffered salinewith 0.05% Tween 20 (TBST) containing 5% skim milk and incubated65with rabbit anti-Trpm5 antibody (1/200 dilution), followed by incubation with horseradish peroxidase (HRP)-conjugated anti-rabbit IgG(#31460, Life Technologies) and subsequent reaction with tyramide
signal ampliﬁcation (TSA)-Alexa Fluor 488 (Life Technologies) as a substrate for peroxidase. After antigen retrieval, the sections were
blocked with TBST containing 5% skim milk and incubated withrabbit anti-Skn-1a antibody (1/300 dilution) or rabbit anti-Dclk1 antibody (1/50 dilution), followed by incubation with Cy3-conjugatedanti-rabbit IgG (#711-165-152, Jackson ImmunoResearch, West
Grove, PA).

Fig. 3. Metabolic phenotypes of Skn-1 KO mice fed a HFD. (a) Changes in body weight under HFD conditions. When fed a HFD for 12 weeks after 4 weeks of age, Skn-1 KO mice exhibited
dramatically reduced body weight compared with their WT littermates (n N 54). (b) Skn-1 KO mice had a lower body fat percentage than their WT littermates, whereas there were no
signiﬁcant differences in muscle mass percentage between the two genotypes (n = 18–26). (c) Skn-1 KO mice had lower tissue weights of epididymal and perirenal WAT (n = 6–12).
(d) Skn-1 KO and WT littermates had similar food intake at 8 weeks of age (n = 8–10). (e) Skn-1 KO mice exhibited increased energy expenditure compared with their WT littermates(n = 4). The data are presented as the mean ± SEM; **p b 0.01 and *p b 0.05. See also Fig. S3.

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2.14. In Situ Hybridization

3.2. Metabolic Phenotypes of Skn-1 KO Mice Fed a Normal Chow Diet

In situ hybridization was performed as previously described(Ishimaru et al., 2005). In brief, fresh-frozen sections (10-μm thick) of
the small intestine, including the duodenum, jejunum, and ileum,
were placed on MAS-coated glass slides (Matsunami Glass, Osaka,
Japan) and ﬁxed in 4% PFA in PBS. Prehybridization (at 58 °C for 1 h), hybridization (at 58 °C, 2 O/N), washing (0.2 × SSC at 58 °C), and development (NBT-BCIP) were performed using a digoxigenin-labeled probe for
Trpm5. Stained images were obtained using a ﬂuorescence microscope(BX51, Olympus, Tokyo, Japan) equipped with a cooled CCD digital camera (DP71, Olympus).

To examine the effect of eliminating type II taste cells and brush cells
in the GI tract on energy homeostasis, we characterized the metabolic
phenotypes of Skn-1 KO mice. Whereas there was no signiﬁcant difference in body weight at birth between the Skn-1 KO and WT littermates(Fig. S2a), Skn-1 KO mice exhibited lower body weight compared totheir WT littermates when weaned at 3 weeks of age (Figs. 2a andS2a). When fed a normal chow diet, Skn-1 KO mice exhibited signiﬁcantly lower body weight than their WT littermates throughout the
13 weeks after weaning (Fig. 2a). For example, the body weights of
the Skn-1 KO and WT littermates at 16 weeks of age were 27.4 ± 0.5 g
and 29.8 ± 0.4 g, respectively. X-ray computed tomography (CT) analysis of body composition revealed that Skn-1 KO mice had a lower
body fat percentage than their WT littermates at approximately
20 weeks of age, whereas there was no signiﬁcant difference in muscle
mass percentage between the two genotypes (Fig. 2b). Among blood
chemical parameters, the concentration of serum total ketone bodies
was signiﬁcantly higher in Skn-1 KO mice than in their WT littermates
under ad libitum feeding and 18-h fasting conditions (Fig. 2c and d).
In addition, some parameters related to lipid metabolism, such as cholesterol and non-esteriﬁed fatty acid (NEFA) levels, tended to be higher
in Skn-1 KO mice compared with their WT littermates (Tables S1 andS2). Collectively, these results demonstrate that lipid metabolism, suchas lipid degradation and β-oxidation of fatty acids, is accelerated in
Skn-1 KO mice.
Despite the lower body weight and body fat percentage of Skn-1 KO
mice, there was no difference in food intake between the Skn-1 KO andWT littermates at 8 weeks of age (Fig. 2e) and in water intake between
the two genotypes (data not shown). Analysis of the metabolic rates ofthe mice revealed that the energy expenditure of Skn-1 KO mice washigher than that of their WT littermates (Fig. 2f). By contrast, there
were no signiﬁcant differences in the respiratory exchange ratio (RER)

2.15. Data Analysis and Statistical Methods
All data are presented as the mean ± SEM. Statistically signiﬁcantdifferences were assessed using Student's t-test.
3. Results
3.1. Brush Cells in the GI Tract Are Abolished in Skn-1 KO MiceWe ﬁrst investigated whether Skn-1a plays important roles in theregulation of cell differentiation in the stomach and small intestine. To
examine the mRNA expression of Skn-1a in the GI tract, we performed
RT-PCR analysis of mRNA from several tissues of WT mice. Skn-1a wasabundantly expressed in the circumvallate papillae, stomach, duodenum, jejunum, and ileum, but it was absent or only faintly expressedin all other tissues examined, including the pancreas, islets ofLangerhans, brain, liver, skeletal muscle, white adipose tissue (WAT),
and brown adipose tissue (BAT) (Fig. 1a). RT-PCR of GAPDH as a control
revealed comparable levels in all the tissues other than islets ofLangerhans and WAT, in which slightly less intense bands were detected (Fig. 1a). To examine the tissue distribution of Skn-1a in the stomach
and intestine at the cellular level, we next performed immunohistochemistry using an anti-Skn-1a antibody. In the stomach, Skn-1a signals
were abundantly detected in the corpus beneath the limiting ridge (Fig.
1b). In the duodenum, jejunum, and ileum, Skn-1a signals were observed in approximately 100 cells per section (Fig. 1b and data notshown). These results suggest that Skn-1a functions as a transcription
factor that regulates cell differentiation in the stomach and intestine
as well as in the taste buds, nasal respiratory epithelium, and main olfactory epithelium (Matsumoto et al., 2011; Ohmoto et al., 2013;
Yamaguchi et al., 2014).
We next examined the expression of Trpm5 in the small intestine. In
situ hybridization analysis using a probe for Trpm5 revealed that Trpm5
signals scattered throughout the small intestine including the duodenum, jejunum, and ileum, of WT mice (Fig. 1c and data not shown), consistent with the previous reports (Bezencon et al., 2008). Intriguingly,
no signals of Trpm5 were observed in Skn-1 KO mice (Fig. 1c). Immunohistochemistry using an anti-Trpm5 antibody conﬁrmed the presence of
the Trpm5 protein in the duodenum of WT mice but the absence of
Trpm5 protein in Skn-1 KO mice (Fig. S1a).
Doublecortin-like kinase 1 (Dclk1) antibody marks brush cells(Gerbe et al., 2009, 2011; Saqui-Salces et al., 2011). Chromogranin A(ChgA) is a marker for enteroendocrine cells (Rindi et al., 2004). GLP1 is an incretin, a peptide hormone, secreted from enteroendocrine L
cells (Wu et al., 2015). Signals of Dclk1 were detected in WT mice but
not in Skn-1 KO mice (Fig. 1d). By contrast, signals of ChgA and GLP-1
were observed in WT and Skn-1 KO mice (Fig. S1a). Finally, we performed double staining using antibodies against Trpm5 and Skn-1a or
Dclk1. Nearly all Trpm5-positive cells were also positive for Skn-1a or
Dclk1 (Figs. 1e and S1b). Taken together, these results demonstratethat brush cells expressing Trpm5 and Dclk1 are abolished in Skn-1 KOmice and that Skn-1a is a crucial transcription factor for generatingbrush cells in the GI tract.

Fig. 4. Enhanced catecholamine secretion in Skn-1 KO mice fed a HFD. (a) Serumtriiodothyronine (T3), tetraiodothyronine (T4), and thyroid stimulating hormone (TSH)
were comparable between Skn-1 KO and WT littermates (n = 7–8). (b) Urinary
epinephrine and dopamine were higher in Skn-1 KO mice than their WT littermates(n = 13–14). The data are presented as the mean ± SEM; *p b 0.05. See also Fig. S4.

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or spontaneous motor activity between the two genotypes (Figs. S2band S2c). These results demonstrate that increased energy expenditurecauses reduced body weight with lower body fat percentage in Skn-1 KO
mice.
Quantitative real-time PCR (qPCR) analysis revealed that the mitochondrial DNA content in the skeletal muscle was signiﬁcantly higher
in Skn-1 KO mice than in their WT littermates (Fig. 2g). Moreover,DNA microarray analysis revealed enhanced expression of genes involved in mitochondrial function, such as uncoupling proteins 3

67(Ucp3), in Skn-1 KO mice (Table 1). These results suggest that energy
expenditure is increased in the skeletal muscle of Skn-1 KO mice.
3.3. Metabolic Phenotypes of Skn-1 KO Mice Fed a HFD
We next examined the metabolic phenotypes of Skn-1 KO mice fed a
HFD. When fed a HFD for N12 weeks after 4 weeks of age, the bodyweight of Skn-1 KO mice was dramatically lower than that of their WT
littermates (Fig. 3a). For example, the body weights of Skn-1 KO and

Fig. 5. Reduced insulin secretion and improved glucose tolerance and insulin resistance in Skn-1 KO mice. (a and b) Blood glucose (a) and plasma insulin (b) after glucose gavage (3.0 mg/gof body weight) when fed a normal chow diet. Blood glucose was comparable between Skn-1 KO and WT littermates for 120 min after administration (n = 21–33) (a). Skn-1 KO mice
exhibited a smaller increase in plasma insulin for 60 min after administration compared with their WT littermates (n = 20–21) (b). (c and d) Blood glucose (c) and plasma insulin (d)after glucose gavage (2.0 mg/g of body weight) of mice fed a HFD. Blood glucose was lower in Skn-1 KO mice than their WT littermates for 120 min after administration (n = 26–30)(c). Plasma insulin was lower in Skn-1 KO mice than their WT littermates before glucose administration (p b 0.05) and tended to be lower at 15 min after administration (p = 0.10)(n = 25–30) (d). (e) Blood glucose after intraperitoneal administration of insulin (0.75 mU/g of body weight) when fed a HFD. Skn-1 KO mice exhibited improved insulin resistancecompared with WT littermates (n = 5). The data are presented as the mean ± SEM; **p b 0.01 and *p b 0.05. See also Fig. S5.

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WT littermates at 16 weeks of age were 30.4 ± 0.9 g and 39.5 ± 0.9 g,
respectively. CT analysis of body composition and measurement of tissue mass revealed that Skn-1 KO mice had a lower body fat percentage,
including epididymal and perirenal white adipose tissues, than their WT
littermates at approximately 20 weeks of age, whereas there were no
signiﬁcant differences in the weight ratios of other tissues, such as muscle, between the two genotypes (Fig. 3b and c and Table S3).
Although there was no signiﬁcant difference in food intake between
the Skn-1 KO and WT littermates at 8 weeks of age (Fig. 3d), energy expenditure was higher in Skn-1 KO mice than in their WT littermates, butspontaneous motor activity was similar between the two genotypes(Fig. 3e and data not shown). In addition, we observed no differencesin food intake or fecal triacylglycerol (TG) between the Skn-1 KO andWT littermates at 15 weeks of age (Figs. S3a and S3b). Fecal energy content was also comparable between the Skn-1 KO and WT littermates(Fig. S3c). These results suggest that increased energy expenditure is
the primary cause of reduced adiposity in Skn-1 KO mice fed a HFD.
Finally, we measured plasma adiponectin, FGF21, insulin, and leptin
in Skn-1 KO and WT littermates fed a HFD (Table S4). Plasma leptin
tended to be lower in Skn-1 KO mice compared with their WT littermates (p = 0.08).

3.4. Catecholamine Secretion is Augmented in Skn-1 KO Mice Fed a HFD
We examined whether the levels of thyroid hormones, including triiodothyronine (T3), tetraiodothyronine (T4), and thyroid stimulating
hormone (TSH), and catecholamines, including norepinephrine, epinephrine, and dopamine, were altered in Skn-1 KO mice fed a HFD.
Serum T3, T4, and TSH were comparable between Skn-1 KO and WT littermates (Fig. 4a). We next measured 24-h urinary excretion of catecholamines. Urinary epinephrine and dopamine were signiﬁcantly
higher and norepinephrine tended to be higher (p = 0.06) in Skn-1
KO mice compared to their WT littermates (Fig. 4b). qPCR analysis revealed that the gene expression of catecholamine biosynthetic enzymes,
including tyrosine hydroxylase (Th), dopa decarboxylase (Ddc), dopamine-β-hydroxylase (Dbh), and phenylethanolamine N-methyltransferase (Pnmt), in adrenal glands was comparable between Skn-1 KO

and their WT littermates (Fig. S4). These results suggest that catecholamines are over-secreted from adrenal glands in Skn-1 KO mice.

3.5. Insulin Secretion is Reduced in Skn-1 KO MiceTo examine carbohydrate metabolism in Skn-1 KO mice, we performed an oral glucose tolerance test (OGTT). Blood glucose concentrations in Skn-1 KO and WT littermates were comparable for 120 min after
oral glucose administration (Fig. 5a). By contrast, the increase in plasma
insulin 60 min after glucose administration was smaller in Skn-1 KOmice than in their WT littermates (Fig. 5b). However, plasma GIP and
GLP-1, which potentiate insulin secretion from pancreatic β-cells (Wuet al., 2015), were comparable between Skn-1 KO and WT littermates
after oral glucose administration (Figs. S5a and S5b).
We next examined the insulin secretion ability of pancreatic β-cells
in Skn-1 KO mice. First, RT-PCR analysis revealed that Skn-1a was not
expressed in the pancreas or islets of Langerhans of WT mice (Fig. 1a).
Second, insulin secretion from isolated islets in response to 25 mM glucose was comparable between Skn-1 KO and WT littermates (Fig. S5c).
Third, in the intraperitoneal glucose tolerance test (IPGTT), blood glucose concentrations were comparable between Skn-1 KO and WT littermates for 120 min after administration (Fig. S5d). Plasma insulin
concentrations were also comparable between Skn-1 KO and WT littermates before and at 15 min after intraperitoneal administration (Fig.
S5e). Collectively, these results demonstrate that insulin secretionfrom pancreatic β-cells is normal in Skn-1 KO mice. Finally, the insulin
tolerance test (ITT) revealed that Skn-1 KO and WT littermates had comparable insulin sensitivity (Fig. S5f).
When fed a HFD, blood glucose was signiﬁcantly lower in Skn-1 KOmice than their WT littermates for 120 min after oral glucose administration (Fig. 5c), demonstrating that impaired glucose tolerance in WT
littermates fed a HFD is improved in Skn-1 KO mice. In addition, plasma
insulin levels were lower in Skn-1 KO mice than in their WT littermates
before glucose administration (p b 0.05) and tended to be lower at
15 min after administration (p = 0.10, Fig. 5d). ITT revealed that theSkn-1 KO mice had improved insulin resistance compared with their
WT littermates (Fig. 5e). Collectively, these results demonstrate that

Fig. 6. Schematic representation of the presumed metabolic pathways originating from brush cells and type II taste cells in the GI tract. Food components and digested nutrients aredetected by the tongue and GI tract. In Skn-1 KO mice, the lack of type II taste cells and brush cells results in increased catecholamine secretion. Lipolysis in WAT, serum levels of total
ketone bodies, and thermogenesis in muscle are increased, whereas insulin secretion from the pancreas is reduced. Consequently, Skn-1 KO mice have reduced body weight withlower body fat percentage due to higher energy expenditure.

S. Ushiama et al. / EBioMedicine 8 (2016) 60–71

the insulin resistance and impaired glucose tolerance observed in WT
mice fed a HFD are ameliorated in Skn-1 KO mice.
4. Discussion
In the present study, we demonstrated that brush cells and type II
taste cells in the GI tract (Matsumoto et al., 2011) are unexpectedly
abolished in Skn-1 KO mice. This ﬁnding indicates that Skn-1 is critical
for the development of Trpm5-expressing chemosensory cells in mice.
The Skn-1 KO mice had reduced body weight with a lower body fat percentage, primarily due to increased energy expenditure. Furthermore,catecholamine secretion was increased in Skn-1 KO, accompanied bydecreased insulin secretion. We propose brain-mediated mechanismsof energy homeostasis originating from brush cells and taste cells in
the GI tract to peripheral tissues including the adrenal glands (Fig. 6).
In detail, taste signals and nutrient stimuli, which are sensed by taste
cells and brush cells in the GI tract are transmitted to the brain via afferent neurons and/or humoral mediators and subsequently conveyed toadrenal glands that secrete catecholamines via efferent sympathetic
nerves. In Skn-1 KO mice, loss of taste and/or nutrient sensing may intensify the activity of the efferent sympathetic nerves, resulting in exaggerated secretion of catecholamines. Based on the network of the gutbrain axis originating from brush cells and taste cells in the GI tract,
the present study suggests an approach to the treatment of obesity.
Skn-1a is expressed in Trpm5-expressing chemosensory cells. Lossof-function of Skn-1a results in the elimination of these cell types(Matsumoto et al., 2011; Ohmoto et al., 2013; Yamaguchi et al., 2014).
In the present study, we demonstrated that Skn-1a is expressed in
Trpm5-expressing brush cells in the GI tract including stomach and intestine. It should be noted that these cells are abolished in the GI tractof Skn-1 KO mice. Genes encoding sweet taste receptors (T1R2 andT1R3) and downstream signaling molecules such as gustducin are
expressed in enteroendocrine L cells that secret GLP-1 (Jang et al.,2007). When glucose was orally administered, the plasma GLP-1 concentration was not augmented in gustducin KO mice compared with
WT mice, which resulted in a lack of a rapid increase in plasma insulinlevels and an impaired glucose tolerance (Jang et al., 2007). We showedthat GLP-1 signals were similar between WT and Skn-1 KO mice in immunohistochemical analyses, consistent with normal incretin release after
oral glucose administration in Skn-1 KO mice. These results demonstratethat Skn-1a is also required for the generation of Trpm5-expressing
brush cells, but not for that of enteroendocrine L cells, in the GI tract.
Trpm5 is expressed not only in the chemosensory cells but also in themouse pancreatic β-cell line MIN6, pancreatic islets of Langerhans, andpancreatic β-cells (Colsoul et al., 2010; Nakagawa et al., 2009; Oya et al.,2011; Prawitt et al., 2003; Yamamoto and Ishimaru, 2013). Trpm5 plays
an important role in the regulation of GSIS from pancreatic β-cells via
two major mechanisms: triggering and amplifying pathways (Brixel etal., 2010; Colsoul et al., 2010; Liman, 2010). Trpm5 KO mice, like Skn-1
KO mice, gain signiﬁcantly less body weight and fat mass and are
more sensitive to insulin than WT mice when fed either a HFD or ahigh-carbohydrate diet (Larsson et al., 2015). T1R3 is the common subunit of sweet (T1R2 + T1R3) and umami (T1R1 + T1R3) taste receptors(Yarmolinsky et al., 2009). When fed a HFD, T1R3 KO mice had reduced
fat mass, in spite of unaltered body weight (Simon et al., 2014). T1R3 isexpressed in the gut, pancreas, and adipose tissue as well as in the
tongue (Yamamoto and Ishimaru, 2013). In contrast, Skn-1a was not
expressed in the pancreas, islets of Langerhans, or brain, suggestingthat the tissue distribution of Skn-1a is more restricted than that of
Trpm5 and T1R3. In this context, Skn-1 KO mice are unique models forinvestigating unexplored mechanisms underlying the gut-brain axis
and whole-body energy metabolism.
Skn-1 KO mice exhibited lower body weight than their WT littermates after weaning when fed either a normal chow diet or a HFD,
whereas body weight was indistinguishable between Skn-1 KO andWT littermates at birth. Notably, the Skn-1 KO mice had much lower

69

body weight than their WT littermates when fed a HFD; the bodyweight gain of Skn-1 KO mice fed a HFD for 12 weeks was nearly equivalent to that of WT mice fed a chow diet during the same period, and theSkn-1 KO mice did not become obese. In Skn-1 KO mice fed a HFD, blood
glucose levels and insulin secretion as well as insulin tolerance were improved, and plasma leptin, an indicator of body fat mass, tended to belower than in their WT littermates. These results demonstrate that
Skn-1 KO mice are resistant to HFD-induced metabolic dysregulation.
The reduced body weight of Skn-1 KO mice was not due to decreasedfood intake or nutrient absorption but to increased energy consumption, which resulted in a lower body fat ratio. Skn-1 KO mice exhibitedslightly but signiﬁcantly higher urinary epinephrine and dopamine, implying an increase in the total amounts of these hormones released in
24 h. Because there was no difference in the expression of the biosynthetic enzymes for catecholamine in the adrenal glands, it is unlikelythat this signiﬁcant increase in catecholamine excretion was caused bycatecholamine overproduction. Because peripheral secretion of catecholamine is induced by activation of the sympathetic nervous system(Watanabe et al., 1988) and Skn-1a is not expressed in the brain, it is
tempting to speculate that the sympathetic nervous system in Skn-1
KO mice is activated by unidentiﬁed signals from the GI tract to thebrain and not alteration of brain function.
In particular, epinephrine and norepinephrine are critical to maintain energy production under hypoglycemia and malnutrition throughlipolysis, ketogenesis (Bahnsen et al., 1984; Steiner et al., 1991), and increased mitochondria in skeletal muscle (Miura et al., 2007). In adipocytes, catecholamines induce lipolysis, but insulin inhibits lipolysis(Nielsen et al., 2014). Consistent with previous reports, the copy numberof mitochondrial DNA and the concentration of serum total ketone bodies
were increased in Skn-1 KO mice, in accordance with an increase inenergy consumption in this model. These results indicate that the appropriate elevation of catecholamine secretion in Skn-1 KO mice may protect
against obesity and metabolic syndrome. Acute psychosocial stress induces changes in catecholamine sensitivity (Strahler et al., 2015). Pheochromocytoma is a tumor arising from adrenomedullary choromafﬁncells that automatically overproduce catecholamines (Lenders et al.,2014). Patients with pheochromocytoma with similar levels of circulatingcatecholamines exhibit variable phenotypes, possibly due in part to differences in hormone sensitivity to the corresponding receptors (Markou etal., 2015). Catecholamine sensitivity in peripheral tissues may be similarly
enhanced by stress signals from the brain in Skn-1 KO mice.
In the OGTT, Skn-1 KO mice exhibited a smaller increase in plasma
insulin compared with their WT littermates, whereas blood glucose
concentrations were indistinguishable between Skn-1 KO and WT littermates. Why did the Skn-1 KO mice exhibit such a reduced increase inplasma insulin despite similar changes in blood glucose? First, plasma
concentrations of incretins, such as GIP and GLP-1, were comparable between Skn-1 KO and WT littermates after oral glucose administration,
which is consistent with the immunohistochemical analyses revealingthat GLP-1 signals were similar between WT and Skn-1 KO mice. Second,
insulin secretion from pancreatic β-cells was normal in Skn-1 KO mice.
Third, Skn-1 KO and WT littermates showed comparable insulin sensitivity in the ITT. By contrast, urinary epinephrine and dopamine weresigniﬁcantly higher in Skn-1 KO mice than their WT littermates. Catecholamines inhibit insulin secretion from pancreatic β-cells (Barth etal., 2007). Overexpression of pancreatic alpha2A-adrenergic receptor
in the diabetic Goto-Kakizaki rat mediates adrenergic suppression of insulin secretion (Rosengren et al., 2009). Higher catecholamine secretion
may inhibit insulin secretion in Skn-1 KO mice. Collectively, our results
demonstrate that enhanced catecholamine secretion in Skn-1 KO miceinduces lipolysis in WAT, accelerates β-oxidation of fatty acids in theliver and skeletal muscle, and, consequently, thermogenesis in skeletal
muscle, and inhibits insulin secretion from the pancreas. A range of diverse effects contribute to resistance to HFD-induced obesity and fueldyshomeostasis (Fig. 6). Further studies are warranted to elucidate themolecular mechanisms underlying the regulation of energy metabolism

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originating from brush cells and type II taste cells in the GI tract, e.g.,
identifying which chemosensory receptors are expressed in the brushcells of the GI tract.
5. Conclusions
In a network of the gut-brain axis, the loss of brush cells and taste
cells in the GI tract alters energy metabolism in Skn-1 KO mice. We
thus propose the concept that taste-receiving cells in the oral cavity
and/or food-borne chemicals-receiving brush cells in the gut are involved in regulation of the body weight and adiposity. The discovery
of food-derived factors that regulate these cells may open new avenues
for the treatment of obesity and diabetes.
Conﬂicts of Interest
The authors declare that they have no conﬂicts of interest.
Author Contributions
S.U., Y.I., and K.A. designed the experiments. S.U., Y.I., M.N., M.Y., andN.W. performed experiments and analyzed the data. C.K. and M.T. guided the experimental methods. N.O., T.A., and H.M. participated in discussions. S.U., Y.I., M.N., H.M., and K.A. wrote the paper.
Acknowledgments
We are grateful to Ms. Taeko Fukuda, Ayako Takahashi, Haruka
Fujihira, and Rieko Imabayahsi for excellent technical assistance. We
thank Dr. Ichiro Matsumoto and Dr. Makoto Ohmoto for backcrossing
Skn-1-deﬁcient mice to C57BL/6J mice. This work was supported inpart by a Grant-in-Aid for Scientiﬁc Research (B) 25292068 to Y.I.;
Grant-in-Aid for challenging Exploratory Research 26242007 to K.A.
from the Ministry of Education, Culture, Sports, Science, and Technologyof Japan; and the Council for Science, Technology and Innovation (CSTI)
Cross-ministerial Strategic Innovation Promotion Program (SIP), “Technologies for creating next-generation agriculture, forestry andﬁsheries.”
Appendix A. Supplementary data
Supplementary data to this article can be found online at http://dx.
doi.org/10.1016/j.ebiom.2016.04.031.
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