   
Catecholamines Facilitate Fuel Expenditure and Protect Against Obesity via a
Novel Network of the Gut-Brain Axis in Transcription Factor Skn-1-deficientMice
Shota Ushiama, Yoshiro Ishimaru, Masataka Narukawa, Misako Yoshioka, Chisayo Kozuka, Naoki Watanabe, Makoto Tsunoda, Naomi Osakabe,
Tomiko Asakura, Hiroaki Masuzaki, Keiko Abe
PII:
DOI:
Reference:

S2352-3964(16)30174-8
doi: 10.1016/j.ebiom.2016.04.031
EBIOM 584

To appear in:

EBioMedicine

Please cite this article as: Ushiama, Shota, Ishimaru, Yoshiro, Narukawa, Masataka, Yoshioka, Misako, Kozuka, Chisayo, Watanabe, Naoki, Tsunoda, Makoto, Osakabe, Naomi, Asakura, Tomiko, Masuzaki, Hiroaki, Abe, Keiko, Catecholamines Facilitate Fuel Expenditure and Protect Against Obesity via a Novel Network of the
Gut-Brain Axis in Transcription Factor Skn-1-deﬁcient Mice, EBioMedicine (2016), doi:
10.1016/j.ebiom.2016.04.031

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Title:T

Catecholamines facilitate fuel expenditure and protect against obesity via a novel

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network of the gut-brain axis in transcription factor Skn-1-deficient miceSC

Authors/Affiliations:Shota Ushiama1, #, Yoshiro Ishimaru1, #, Masataka Narukawa1, #, Misako Yoshioka1,

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Chisayo Kozuka2, Naoki Watanabe3, Makoto Tsunoda4, Naomi Osakabe3, Tomiko
Asakura1, Hiroaki Masuzaki2, Keiko Abe1, 5

1ED

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
2

PT

Division of Endocrinology, Diabetes and Metabolism, Hematology, Rheumatology(Second Department of Internal Medicine), Graduate School of Medicine, University ofCE

the Ryukyus, 207 Uehara, Nishihara, Okinawa 903-0215, Japan
3

AC

Department of Bio-science and Engineering, Shibaura Institute of Technology, 307

Fukasaku, Minuma-ku, Saitama 337-8570, Japan.
4

Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo,

Bunkyo-ku, Tokyo 113-0033, Japan
5

Kanagawa Academy of Science and Technology, Takatsu-ku, Kawasaki-shi, Kanagawa,Japan

Contact:
Address: Department of Applied Biological Chemistry, Graduate School of Agriculturaland Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657,
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Japan;

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Tel: +81-3-5841-1878, Fax: +81-3-5841-1879

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E-mail: ayishi@mail.ecc.u-tokyo.ac.jp (Y.I.) and aka7308@mail.ecc.u-tokyo.ac.jp (K.A.)

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Additional Footnotes: # These authors contributed equally to this work.

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Key words:

energy metabolism, brush cells, catecholamine, insulin

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Running title:

Abbreviations:PT

Effect of Skn-1 gene deletion on energy homeostasis via the gut-brain axis

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BAT, brown adipose tissue; ChgA, chromogranin A; CT, computed tomography; Dbh,

GI,

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dopamine-β-hydroxylase; Dclk1, doublecortin-like kinase 1; Ddc, dopa decarboxylase;
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,
solitary chemosensory 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.
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Highlights

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Skn-1a is a crucial transcription factor for generating brush cells and type II taste cells in

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the gastrointestinal tract.

body fat due to elevated energy expenditure.

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Despite unaltered food intake, Skn-1 KO mice have reduced body weight with lower

Urinary excretion of catecholamines was elevated in Skn-1 KO mice, suggestingMA
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brain-mediated energy homeostatic pathways.

Research in context

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Taste signals and nutrient stimuli sensed by the gastrointestinal tract are transmitted tothe brain to regulate feeding behavior and energy homeostasis along the gut-brain axis.

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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 weightCEand adiposity via the brain. The discovery of food-derived factors that regulate theseACcells may open new avenues for the treatment of obesity and diabetes.

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SummaryT

Taste signals and nutrient stimuli sensed by the gastrointestinal tract are transmitted to

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the brain to regulate feeding behavior and energy homeostasis. This system is referred
to as the gut-brain axis. Here we show that both brush cells and type II taste cells areSCeliminated 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

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body fat due to increased energy expenditure. In this model, 24-hr urinary excretion of
catecholamines was significantly elevated, accompanied by increased fatty acid-oxidation and fuel dissipation in skeletal muscle and impaired insulin secretion driven

EDby glucose. These results suggest the existence of brain-mediated energy homeostatic
pathways originating from brush cells and type II taste cells in the gastrointestinal tractPTand ending in peripheral tissues, including the adrenal glands. The discovery offood-derived factors that regulate these cells may open new avenues the treatment ofACCEobesity and diabetes.

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Introduction

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Taste signals and nutrient stimuli sensed by the gastrointestinal (GI) tract are

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transmitted to the central nervous system, including the nucleus of the solitary tract andhypothalamus, via afferent neurons and humoral mediators, thereby controlling feedingSCbehavior and energy homeostasis. This system is referred to as the gut-brain axis
(Cummings and Overduin, 2007; Furness, 2012). This information is subsequently

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conveyed from the brain to peripheral tissues via efferent sympathetic andparasympathetic nerves including preganglionic sympathetic splanchnic input to theadrenal glands. For example, gut incretins such as glucose-dependent insulinotropicED

peptide (GIP) and glucagon-like peptide-1 (GLP-1), which are released from theintestinal enteroendocrine cells in response to nutrient ingestion, potentiate

PTglucose-stimulated insulin secretion (GSIS) from pancreatic β-cells (Wu et al., 2015).
GLP-1 also activates the GLP-1 receptors expressed on vagal afferent nerve terminals inCEthe portal vein and in the brain across the blood-brain barrier, which is involved in theACregulation of appetite (Katsurada et al., 2014; Kinzig et al., 2002; Vahl et al., 2007).
Gavage of agonists for bitter taste receptor increases food intake via the secretion ofghrelin, a hormone that potentiates hunger sensation (Janssen et al., 2011).
The Skn-1 (also known as Pou2f3) gene, which encodes the POU homeodomaintranscription factor, was originally identified as a regulator of the differentiation of
epidermal keratinocytes (Andersen et al., 1993; Andersen et al., 1997). We previously
reported that Skn-1a is expressed in sweet, 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 ofelectrophysiological and behavioral responses to sweet, umami, and bitter tastes. Thus,
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Skn-1a is critical for generating type II taste cells. Skn-1a is also required for theTgeneration of Trpm5-expressing solitary chemosensory cells (SCCs) in the nasal

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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 theSCstomach, but not in the brain (Yukawa et al., 1993).

There are four major cell types in the small intestine: enterocytes and Goblet, Paneth,

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and enteroendocrine cells (van der Flier and Clevers, 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

EDare supposed to be chemosensory cells (Young, 2011) and express transient receptor
potential melastatin 5 (Trpm5), Doublecortin-like kinase 1 (Dclk1), and cholinePTacetyltransferase (Bezencon et al., 2008; Gerbe et al., 2009; Gerbe et al., 2011;
Saqui-Salces et al., 2011; Schütz B et al., 2015). The mechanisms of differentiation andCEthe function of brush cells remain elusive.

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In the present study, we found that Skn-1a regulates differentiation of
Trpm5-expressing brush cells in the GI tract. Skn-1 KO mice exhibited reduced bodyweight 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 andtaste cells in the GI tract and signaling to peripheral tissues including the adrenal glands,
via the brain.
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Material and Methods

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Experimental Animals

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All animal experiments were approved by the Animal Care and Use Committee at The
University of Tokyo. Skn-1/Pou2f3-deficient mice with a mixed 129 x C57BL/6J

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background were generated as previously described (Matsumoto et al., 2011) and thenbackcrossed to C57BL/6J for more than 10 generations. Male Skn-1 KO mice and theirMA
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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

EDof age for more than 12 weeks to induce dietary obesity. Mice were maintained in a roomwith a constant temperature of 22±1°C under a 12-hr light-dark cycle (lights on at 8 a.m.).

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Tissue samples were collected from 12- to 16-week-old mice that were fasted for 18 hr.

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X-ray Computed Tomography (CT) Scan Analysis

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Mice older than 16 weeks were anesthetized using an isoflurane nebulizer (Muromachi
Kikai Co. Ltd., Tokyo, Japan); a Latheta LCT-200 CT instrument (Aloka-Hitachi LCT-200,
Tokyo, Japan) was then used to scan abdominal fat and muscle mass and extract
thighbone density.

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-hr fasting. Serum chemical parametersand hormones (PTH, T3, and T4) were analyzed by Nagahama Life Science Laboratory(Shiga, Japan). Plasma insulin and leptin were measured using the Luminex 200™
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System (Luminex, Austin, TX, USA) by GeneticLab Co., Ltd. (Sapporo, Japan). PlasmaTadiponectin and FGF21 were measured using mouse adiponectin ELISA (BioVender,

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Brno, Czech Republic) and mouse and rat FGF-21 ELISA (BioVender), respectively.

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Indirect Calorimetry

Indirect calorimetry was performed on 20- to 28-week-old mice using an indirect

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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 byED

Tschop et al. (Tschop et al., 2011) or with an indirect calorimeter (MK-5000RQ,
Muromachi Kikai Co. Ltd.) as previously described (Watanabe et al., 2014). The

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respiratory exchange ratio (RER) was calculated as the molar ratio of VCO2/VO2. Energy

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VO2.

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expenditure (kcal per hour) was calculated as heat (kcal/h) = (3.815 + 1.232 × RER) ×

Mitochondrial DNA Copy Number
To measure the mitochondrial DNA copy number, the ratio of mitochondrial DNA togenomic DNA was calculated by a previously described method (Watanabe et al., 2014).
In brief, total DNA was extracted from the gastrocnemius of 14- to 19-week-old mice thatwere fasted for 16 hr using a QIAamp DNA mini kit (QIAGEN Ltd., Tokyo, Japan) andused 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
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mitochondrial DNA copy number is presented relative to nuclear DNA followingTamplification of the mitochondrial gene region (cytochrome b vs. the nuclear

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endogenous control region, β-actin).

SCDNA Microarray

DNA microarray analysis using total RNA extracted from the gastrocnemius of 12- toMANU

16-week-old mice that were fasted for 18 hr was performed as previously described(Nakai et al., 2008) with minor modifications. In brief, Total RNA samples (100 ng each)were prepared and processed for microarray analysis using a 3’ IVT Express kit and
GeneChip® Mouse Genome 430 2.0 Array (Affymetrix, Santa Clara, CA, USA)ED

according to the manufacturer’s standard protocols. The raw microarray data (CEL files)

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were quantified by the Factor Analysis for Robust Microarray Summarization (FARMS)(Hochreiter et al., 2006) using the statistical language R (Team and Computing, 2005)

CE

and Bioconductor (Gentleman et al., 2004). To identify differentially expressed genes,ACthe 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).

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 the9ACCEPTED MANUSCRIPT

Wako triglyceride E-Test (Wako Pure Chemical Industries, Ltd., Osaka, Japan). The total

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energy content of feces collected for 8 weeks was measured by bomb calorimetry.

Measurement of Urine Catecholamines

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Twenty-four-hour urine (for catecholamine measurements) was collected into collection
vials from 11- to 12-week-old mice individually housed in metabolic cages. To avoid

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spontaneous oxidation of catecholamine, 60 μL of hydrochloric acid (6 mol/L) was added
to the collection vials (Moreira-Rodrigues et al., 2012). The collected urine was purified
using MonoSpin PBA (GL Sciences, Tokyo, Japan) and urine catecholamines were
measured by HPLC as previously described (Tsunoda et al., 2011). In brief,ED

Chromatography was performed using an Inertsil ODS-4 column (5 μm, 250 x 20 mm i.d.,PT

GL Science) with acetate-citrate buffer at a flow rate of 0.5 mL/min and detected byelectrochemical detection. The concentration of urine creatinine was measured using aAC

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LabAssay™ Creatinine kit (Wako Pure Chemical Industries, Ltd.).

qRT-PCR in Adrenal Glands
Total RNA was extracted from adrenal glands using RNeasy mini-columns (Qiagen,
Venlo, Netherlands), followed by genomic DNA 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
of mRNA 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 Assays, Applied Biosystems). The TaqMan probe IDs were as follows:
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tyrosinehydroxylase

Mm00447557_m1),

dopamine

β-hydroxylase

dopa

decarboxylase

(Dbh;

(Ddc;

Mm00460472_m1),

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Mm00516688_m1),

(Th;

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phenylethanolamine N-methyltransferase (Pnmt; Mm00476993_m1), and β-actin(Mm00607939_s1). The delta-delta method was used for relative quantification (Livak

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and Schmittgen, 2001).

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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 measurementEDof plasma active GLP-1; 2.0 mg/g of body weight for measurement of blood glucose andplasma insulin in mice fed a HFD) was performed on 16-week-old WT and Skn-1 KOPTmice at 10 a.m. after 18-hr fasting (n=20-30 animals per group) using a syringe attached
to a feeding needle (KN-349-M1, Natsume Seisakusho Co. Ltd., Tokyo, Japan) insertedCEthrough the mouth into the stomach. For IPGTT or ITT, mice were fasted for 18 or 4 hours,ACthen injected intraperitoneally with glucose (1.5 mg/g of body weight) or insulin (0.75mU/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, tail blood 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 (time 0) and 15, 30, and60 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
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ACCEPTED MANUSCRIPTGIP (Total) ELISA Kit (EZRMGIP-55K, Merck Millipore), and GLP-1 (Active) ELISA Kit

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( AKMGP-011, Shibayagi Co. Ltd., Gunma, Japan), respectively.

RT-PCRSCFirst-strand cDNAs from 11 different tissues except the circumvallate papillae (Genostaff,
Tokyo, Japan) were used for RT-PCR. For the circumvallate papillae, total RNA wasMA
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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 volume of PCR productsED

for Skn-1a loaded on the agarose gel was approximately normalized using the PCR

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product for GAPDH.

Islet Isolation and In Vitro Insulin Release from Mouse Islets

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Isolated pancreatic islets were prepared by liberase (Roche Diagnostics, Basel,

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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 toexperiments, 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
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Krebs-Ringer buffer containing 25 mM glucose (high glucose) for 30 min. The

Tsupernatants from the second low-glucose solution and from the high-glucose solution

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were collected 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

Immunohistochemistry

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Biological Science).

Rabbits were immunized with peptides corresponding to residues 1140-1153(C+PASARDREYLESGL)

of

Trpm5

(OperonBiotechnologies,

Tokyo,

Japan).

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Preincubation of the antibody with peptide antigen (10 ng/ml) abolished the brush cellstaining, confirming the specificity of the antibody (data not shown). The other primaryPT

antibodies used were as follows: rabbit anti-Skn-1a antibody (#sc-330, Santa Cruz
Biotechnology, Santa Cruz, CA), rabbit anti-Dclk1 antibody (#AP7219b, Abgent, San

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Diego, CA), rabbit anti-ChgA antibody (#20085, ImmunoStar, Hudson, WI), and goatAC

anti-GLP-1 antibody (#sc-7782, Santa Cruz Biotechnology). Immunohistochemistry was
performed as previously described (Ishimaru et al., 2006). In brief, Fresh-frozen sections(10-μm thick) were fixed in 4% paraformaldehyde (PFA), permeabilized with ice-coldmethanol, blocked with phosphate-buffered saline (PBS) containing 5% skim milk, andincubated with rabbit anti-Skn-1a antibody (1/1,000 dilution), rabbit anti-Trpm5 antibody(1/200 dilution), rabbit anti-Dclk1 antibody (1/50 dilution), or rabbit anti-ChgA antibody(1/200 dilution), followed by incubation with Alexa 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 anti-GLP-1 antibody
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ACCEPTED MANUSCRIPT(1/150 dilution) followed by incubation with Alexa Fluor 555-conjugated anti-goat IgG

T(#A21432, Life Technologies).

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For double staining, fresh-frozen sections (10-μm thick) were fixed with 4% PFA andpermeabilized with ice-cold methanol followed by antigen retrieval. The sections wereSCthen blocked with Tris-buffered saline with 0.05% Tween 20 (TBST) containing 5% skim
milk and incubated with rabbit anti-Trpm5 antibody (1/200 dilution), followed byMA
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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, theEDsections were blocked with TBST containing 5% skim milk and incubated with rabbit
anti-Skn-1a antibody (1/300 dilution) or rabbit anti-Dclk1 antibody (1/50 dilution),PTfollowed by incubation with Cy3-conjugated anti-rabbit IgG (#711-165-152, JacksonCE

ImmunoResearch, West Grove, PA).

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In situ Hybridization

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 hour), hybridization(at 58°C, 2 O/N), washing (0.2x SSC at 58°C), and development (NBT-BCIP) were
performed using a digoxigenin-labeled probe for Trpm5. Stained images were obtainedusing a fluorescence microscope (BX51, Olympus, Tokyo, Japan) equipped with acooled CCD digital camera (DP71, Olympus).

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Data Analysis and Statistical Methods

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All data are presented as the mean ± SEM. Statistically significant differences were

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assessed using Student’s t-test.

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Results

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Brush cells in the GI tract are abolished in Skn-1 KO mice

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We first investigated whether Skn-1a plays important roles in the regulation of celldifferentiation in the stomach and small intestine. To examine the mRNA expression ofSC

Skn-1a in the GI tract, we performed RT-PCR analysis of mRNA from several tissues of
WT mice. Skn-1a was abundantly expressed in the circumvallate papillae, stomach,

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duodenum, jejunum, and ileum, but it was absent or only faintly expressed in all othertissues examined, including the pancreas, islets of Langerhans, brain, liver, skeletal
muscle, white adipose tissue (WAT), and brown adipose tissue (BAT) (Fig. 1a). RT-PCR

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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

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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,

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Skn-1a signals were abundantly detected in the corpus beneath the limiting ridge (Fig.

AC1b). In the duodenum, jejunum, and ileum, Skn-1a signals were observed inapproximately 100 cells per section (Fig. 1b and data not shown). These results suggestthat Skn-1a functions as a transcription factor that regulates cell differentiation in thestomach and intestine as well as in the taste buds, nasal respiratory epithelium, andmain 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 thesmall intestine including the duodenum, jejunum, and ileum, of WT mice (Fig. 1c anddata not shown), consistent with the previous reports (Bezencon et al., 2008). Intriguingly,
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no signals of Trpm5 were observed in Skn-1 KO mice (Fig. 1c). ImmunohistochemistryTusing an anti-Trpm5 antibody confirmed the presence of the Trpm5 protein in the

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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;

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Gerbe et al., 2011; Saqui-Salces et al., 2011). Chromogranin A (ChgA) is a marker forenteroendocrine cells (Rindi et al., 2004). GLP-1 is an incretin, a peptide hormone,MA
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secreted from enteroendocrine L cells (Wu et al., 2015). Signals of Dclk1 were detectedin 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 doubleED

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, thesePT

results demonstrate that brush cells expressing Trpm5 and Dclk1 are abolished in Skn-1

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GI tract.

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KO mice and that Skn-1a is a crucial transcription factor for generating brush cells in the

Metabolic phenotypes of Skn-1 KO mice fed a normal chow dietTo examine the effect of eliminating type II taste cells and brush cells in the GI tract onenergy 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 comparedto their WT littermates when weaned at 3 weeks of age (Figs. 2a and S2a). When fed anormal 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 bodyweights of the Skn-1 KO and WT littermates at 16 weeks of age were 27.4 ± 0.5 g and
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29.8 ± 0.4 g, respectively. X-ray computed tomography (CT) analysis of bodyTcomposition revealed that Skn-1 KO mice had a lower body fat percentage than their WT

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littermates at approximately 20 weeks of age, whereas there was no significant
difference in muscle mass percentage between the two genotypes (Fig. 2b). AmongSC

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 feedingMA
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and 18-hr fasting conditions (Figs. 2c and 2d). In addition, some parameters related tolipid 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 and S2).
Collectively, these results demonstrate that lipid metabolism, such as lipid degradationEDand β-oxidation of fatty acids, is accelerated in Skn-1 KO mice.

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Despite the lower body weight and body fat percentage of Skn-1 KO mice, there wasno difference in food intake between the Skn-1 KO and WT littermates at 8 weeks of age

CE(Fig. 2e) and in water intake between the two genotypes (data not shown). Analysis of theACmetabolic rates of the 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) or spontaneous motor activity
between the two genotypes (Figs. S2b and S2c). These results demonstrate that
increased energy expenditure causes 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 expressionof genes involved in mitochondrial function, such as uncoupling proteins 3 (Ucp3), in
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Skn-1 KO mice (Table 1). These results suggest that energy expenditure is increased in

Metabolic phenotypes of Skn-1 KO mice fed a HFD

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the skeletal muscle of Skn-1 KO mice.

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We next examined the metabolic phenotypes of Skn-1 KO mice fed a HFD. When fed
a HFD for more than 12 weeks after 4 weeks of age, the body weight of Skn-1 KO miceMA
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was dramatically lower than that of their WT littermates (Fig. 3a). For example, the bodyweights of Skn-1 KO and 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 massED

revealed that Skn-1 KO mice had a lower body fat percentage, including epididymal andperirenal white adipose tissues, than their WT littermates at approximately 20 weeks ofPTage, whereas there were no significant differences in the weight ratios of other tissues,
such as muscle, between the two genotypes (Figs. 3b and 3c and Table S3).

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Although there was no significant difference in food intake between the Skn-1 KO andAC

WT littermates at 8 weeks of age (Fig. 3d), energy expenditure was higher in Skn-1 KOmice than in their WT littermates, but spontaneous 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 and WT 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 KOmice fed a HFD.
Finally, we measured plasma adiponectin, FGF21, insulin, and leptin in Skn-1 KO andWT littermates fed a HFD (Table S4). Plasma leptin tended to be lower in Skn-1 KO mice
19

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compared with their WT littermates (p=0.08).

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Catecholamine secretion is augmented in Skn-1 KO mice fed a HFD

We examined whether the levels of thyroid hormones, including triiodothyronine (T3),SC

tetraiodothyronine (T4), and thyroid stimulating hormone (TSH), and catecholamines,
including norepinephrine, epinephrine, and dopamine, were altered in Skn-1 KO miceMA
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fed a HFD. Serum T3, T4, and TSH were comparable between Skn-1 KO and WT
littermates (Fig. 4a). We next measured 24-hr urinary excretion of catecholamines.
Urinary epinephrine and dopamine were significantly higher and norepinephrine tendedED

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,
tyrosinehydroxylase

(Th),

dopa

decarboxylase(Ddc),

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including

dopamine-β-hydroxylase (Dbh), and phenylethanolamine N-methyltransferase (Pnmt),

CEin adrenal glands was comparable between Skn-1 KO and their WT littermates (Fig. S4).

AC

These results suggest that catecholamines are over-secreted from adrenal glands in
Skn-1 KO mice.

Insulin secretion is reduced in Skn-1 KO mice
To 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 KO mice than in their WT littermates (Fig. 5b). However, plasma GIP and GLP-1,
which potentiate insulin secretion from pancreatic β-cells (Wu et al., 2015), were20

ACCEPTED MANUSCRIPT

comparable between Skn-1 KO and WT littermates after oral glucose administration

T(Figs. S5a and S5b).

RIP

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 isletsSCof 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.

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S5c). Third, in the intraperitoneal glucose tolerance test (IPGTT), blood glucose
concentrations were comparable between Skn-1 KO and WT littermates for 120 min afteradministration (Fig. S5d). Plasma insulin concentrations were also comparable betweenED

Skn-1 KO and WT littermates before and at 15 min after intraperitoneal administration(Fig. S5e). Collectively, these results demonstrate that insulin secretion from pancreaticPTβ-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).

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When fed a HFD, blood glucose was significantly lower in Skn-1 KO mice than theirAC

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<0.05) and tended to be lower at 15 min afteradministration (p=0.10, Fig. 5d). ITT revealed that the Skn-1 KO mice had improved
insulin resistance compared with their WT littermates (Fig. 5e). Collectively, these results
demonstrate that the insulin resistance and impaired glucose tolerance observed in WT
mice fed a HFD are ameliorated in Skn-1 KO mice.

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Discussion

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In the present study, we demonstrated that brush cells and type II taste cells in the GI

RIP

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 chemosensorySC

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

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secretion was increased in Skn-1 KO, accompanied by decreased insulin secretion. We
propose brain-mediated mechanisms of energy homeostasis originating from brush cells
and taste cells in the GI tract to peripheral tissues including the adrenal glands (Fig. 6). InED

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 mediatorsPTand subsequently conveyed to adrenal glands that secrete catecholamines via efferent
sympathetic nerves. In Skn-1 KO mice, loss of taste and/or nutrient sensing may

CEintensify the activity of the efferent sympathetic nerves, resulting in exaggerated

ACsecretion of catecholamines. Based on the network of the gut-brain axis originating frombrush cells and taste cells in the GI tract, the present study suggests an approach to thetreatment of obesity.
Skn-1a is expressed in Trpm5-expressing chemosensory cells. Loss-of-function of
Skn-1a results in the elimination of these cell types (Matsumoto et al., 2011; Ohmoto etal., 2013; Yamaguchi et al., 2014). In the present study, we demonstrated that Skn-1a isexpressed in Trpm5-expressing brush cells in the GI tract including stomach andintestine. It should be noted that these cells are abolished in the GI tract of Skn-1 KO
mice. Genes encoding sweet taste receptors (T1R2 and T1R3) and downstream signaling
molecules such as gustducin are expressed in enteroendocrine L cells that secret GLP-1

22

ACCEPTED MANUSCRIPT(Jang et al., 2007). When glucose was orally administered, the plasma GLP-1 concentrationT

was not augmented in gustducin KO mice compared with WT mice, which resulted in a lackRI
Pof a rapid increase in plasma insulin levels and an impaired glucose tolerance (Jang et al.,2007). We showed that GLP-1 signals were similar between WT and Skn-1 KO mice inSCimmunohistochemical analyses, consistent with normal incretin release after oral
glucose administration in Skn-1 KO mice. These results demonstrate that Skn-1a is alsoMA
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required for the generation of Trpm5-expressing brush cells, but not for enteroendocrine L
cells, in the GI tract.

Trpm5 is expressed not only in the chemosensory cells but also in the mouseEDpancreatic β-cell line MIN6, pancreatic islets of Langerhans, and pancreatic β–cells(Colsoul et al., 2010; Nakagawa et al., 2009; Oya et al., 2011; Prawitt et al., 2003;

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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

CE(Brixel et al., 2010; Colsoul et al., 2010; Liman, 2010). Trpm5 KO mice, like Skn-1 KOACmice, gain significantly less body weight and fat mass and are more sensitive to insulin
than WT mice when fed either a HFD or a high-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 is expressed in thegut, 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, orbrain, suggesting that 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 for investigating
unexplored mechanisms underlying the gut-brain axis and whole-body energy
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metabolism.

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Skn-1 KO mice exhibited lower body weight than their WT littermates after weaning

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when fed either a normal chow diet or a HFD, whereas body weight wasindistinguishable between Skn-1 KO and WT littermates at birth. Notably, the Skn-1 KOSCmice had much lower 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 ofMA
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WT mice fed a chow diet during the same period, and the Skn-1 KO mice did not become
obese. In Skn-1 KO mice fed a HFD, blood glucose levels and insulin secretion as wellas insulin tolerance were improved, and plasma leptin, an indicator of body fat mass,ED

tended to be lower than in their WT littermates. These results demonstrate that Skn-1 KOmice are resistant to HFD-induced metabolic dysregulation.

PT

The reduced body weight of Skn-1 KO mice was not due to decreased food intake or
nutrient absorption but to increased energy consumption, which resulted in a lower bodyCE

fat ratio. Skn-1 KO mice exhibited slightly but significantly higher urinary epinephrine andAC

dopamine, implying an increase in the total amounts of these hormones released in 24
hours. Because there was no difference in the expression of the biosynthetic enzymes
for catecholamine in the adrenal glands, it is unlikely that this significant increase incatecholamine excretion was caused by catecholamine 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 activatedby unidentified signals from the GI tract to the brain and not alteration of brain function.
In particular, epinephrine and norepinephrine are critical to maintain energy production
under hypoglycemia and malnutrition through lipolysis, ketogenesis (Bahnsen et al.,24

ACCEPTED MANUSCRIPT

1984; Steiner et al., 1991), and increased mitochondria in skeletal muscle (Miura et al.,T2007). In adipocytes, catecholamines induce lipolysis, but insulin inhibits lipolysis

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P(Nielsen et al., 2014). Consistent with previous reports, the copy number ofmitochondrial DNA and the concentration of serum total ketone bodies were increased inSC

Skn-1 KO mice, in accordance with an increase in energy consumption in this model.
These results indicate that the appropriate elevation of catecholamine secretion in Skn-1

MANU

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 choromaffin cells thatED

automatically overproduce catecholamines (Lenders et al., 2014). Patients withpheochromocytoma with similar levels of circulating catecholamines exhibit variablePTphenotypes, possibly due in part to differences in hormone sensitivity to thecorresponding receptors (Markou et al., 2015). Catecholamine sensitivity in peripheralCEtissues may be similarly enhanced by stress signals from the brain in Skn-1 KO mice.

AC

In the OGTT, Skn-1 KO mice exhibited a smaller increase in plasma insulin comparedwith 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 in plasma 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 revealing that 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 were significantly
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higher in Skn-1 KO mice than their WT littermates. Catecholamines inhibit insulinTsecretion from pancreatic β-cells (Barth et al., 2007). Overexpression of pancreatic

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alpha2A-adrenergic receptor in the diabetic Goto-Kakizaki rat mediates adrenergic
suppression of insulin secretion (Rosengren et al., 2009). Higher catecholamineSCsecretion may inhibit insulin secretion in Skn-1 KO mice. Collectively, our results
demonstrate that enhanced catecholamine secretion in Skn-1 KO mice induces lipolysis

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in WAT, accelerates β-oxidation of fatty acids in the liver and skeletal muscle, and,
consequently, thermogenesis in skeletal muscle, and inhibits insulin secretion from thepancreas. A range of diverse effects contribute to resistance to HFD-induced obesity andEDfuel dyshomeostasis (Fig. 6). Further studies are warranted to elucidate the molecularmechanisms underlying the regulation of energy metabolism originating from brush cells

PTand type II taste cells in the GI tract, e.g., identifying which chemosensory receptors areACConclusions

CE

expressed in the brush cells of the GI tract.

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 thattaste-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 offood-derived factors that regulate these cells may open new avenues for the treatment ofobesity and diabetes.

Conflicts of Interest
The authors declare that they have no conflicts of interest.
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Author Contributions

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PS.U., Y.I., and K.A. designed the experiments. S.U., Y.I., M.N., M.Y., and N.W. performedexperiments and analyzed the data. C.K. and M.T. guided the experimental methods.

SC

N.O., T.A., and H.M. participated in discussions. S.U., Y.I., M.N., H.M., and K.A. wrote theMA
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paper.

Acknowledgments

We are grateful to Ms. Taeko Fukuda, Haruka Fujihira, and Rieko Imabayahsi forEDexcellent 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 inPTpart by a Grant-in-Aid for Scientific Research (B) 25292068 to Y.I.; Grant-in-Aid forchallenging Exploratory Research 25560046 to K.A. from the Ministry of Education,

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Culture, Sports, Science, and Technology of Japan; and the Council for Science,AC

Technology and Innovation (CSTI) Cross-ministerial Strategic Innovation Promotion
Program (SIP), “Technologies for creating next-generation agriculture, forestry andfisheries.”

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Figure Legends

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Fig. 1. Brush Cells Are Eliminated in the GI Tract of Skn-1 KO Mice

RIP(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 butSCabsent 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

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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 ofED

the area enclosed by the dashed box.

PT(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).

CE(d) Immunostaining revealed Dclk1 signals in WT mice (upper panel) but not Skn-1 KOmice (lower panel). The arrowheads indicate signals.

AC(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.

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>75). Also see Fig. S2A.
(b) Skn-1 KO mice had a lower body fat percentage than their WT littermates, whereas

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there was no significant difference in muscle mass percentage between the twoTgenotypes (n=10-19).

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P(c and d) Serum levels of total ketone bodies were higher in Skn-1 KO mice than their WT
littermates under ad libitum feeding (n=18-28) (c) and 18-hr fasting conditions (n=13) (d).

SC(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 WTMA
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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 areED

presented as the mean ± SEM; **p<0.01, *p<0.05. See also Fig. S2.

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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

CEweeks of age, Skn-1 KO mice exhibited dramatically reduced body weight comparedACwith their WT littermates (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<0.01, *p<0.05. See
also Fig. S3.

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Fig. 4. Enhanced Catecholamine Secretion in Skn-1 KO Mice Fed a HFDT(a) Serum triiodothyronine (T3), tetraiodothyronine (T4), and thyroid stimulating hormone

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P(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 WTSClittermates (n=13–14). The data are presented as the mean ± SEM; *p<0.05. See also

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Fig. S4.

Fig. 5. Reduced Insulin Secretion and Improved Glucose Tolerance and Insulin
Resistance in Skn-1 KO MiceED

(a and b) Blood glucose (a) and plasma insulin (b) after glucose gavage (3.0 mg/g ofbody weight) when fed a normal chow diet. Blood glucose was comparable between

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Skn-1 KO and WT littermates for 120 min after administration (n=21–33) (a). Skn-1 KOmice exhibited a smaller increase in plasma insulin for 60 min after administrationCEcompared with their WT littermates (n=20-21) (b).

AC(c and d) Blood glucose (c) and plasma insulin (d) after glucose gavage (2.0 mg/g ofbody 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<0.05) andtended 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 bodyweight) 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<0.01, *p<0.05. See also Fig. S5.

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Fig. 6. Schematic representation of the presumed metabolic pathways originating fromT

brush cells and type II taste cells in the GI tract

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Food components and digested nutrients are detected by the tongue and GI tract. In
Skn-1 KO mice, the lack of type II taste cells and brush cells results in increasedSC

catecholamine secretion. Lipolysis in WAT, serum levels of total ketone bodies, andthermogenesis in muscle are increased, whereas insulin secretion from the pancreas isMA
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reduced. Consequently, Skn-1 KO mice have reduced body weight with lower body fat

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percentage due to higher energy expenditure.

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stromal cells. Gene 133, 163-169.

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b

**
**

6
**

4 **
WT
KO

2
0

Ad libc
60

Fasting

d
3000

2000

PT

1000

0

CE

WAT

11
10
9
8
7
6
5
4
3
2
1
0

g

*
mtDNA/nDNA ratio

AC
Energy expenditure(kcal/kg BW)

100

*

e

*

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T-KB (μmol/L)

T-KB (μmol/L)

*

0

f

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Age (week-old)

WT
KO

T

**

**

9.6
8.8
8
7.2
6.4
5.6
4.8
4
3.2
2.4
1.6
0.8
0

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**

8

** **

** **

Tissue weight (g/g BW)

Body weight (g)10** ** **

SC

12

Food intake (g/day)

a

*

0

Figure 2 Ushiama et al.

41

11
10
9
8
7
6
5
4
3
2
1
0

*

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ACCEPTED MANUSCRIPT45

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Gene Name

Probe ID

ABCD2

ATP-binding

1419748_at

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Gene Symbol

T

Table 1 Up-regulation genes related to mitochondrial function
in skeletal muscle

cassette,

Ociad2

Pptc7

OCIA domain

1435917_at

containing 2

MA
NU(ALD), member 2

PTC7 protein
phosphatasehomolog (S.
cerevisiae)

4930402E16Rik

RIKEN

1455958_s_at

1459869_x_at

6Rik gene

ED

cDNA4930402E1

RIKEN cDNAPT

8430408G22Rik

1433837_at

8430408G22 gene
TBC1 domainCE

Tbc1d151416062_at

family, member 15

Xaf1

XIAP associated1443698_atACfactor 1

Col4a3bp

collagen, type IV,

1420384_atalpha 3
(Goodpasture
antigen) binding
proteinDsp

desmoplakin1435493_at,
1435494_s_at

DlaT

SC

sub-family D

dihydrolipoamide

1426264_at,

S-acetyltransferase

1426265_x_at(E2 component of
pyruvate
dehydrogenase
complex)46

ACCEPTED MANUSCRIPT

Fdx1

ferredoxin 1

1449108_at

Gpam

glycerol-3-phosph1419499_atTate acyltransferase,

Hk2

hexokinase 21422612_at

Coq10bcoenzyme Q101460510_a_at

RIP

mitochondrial

Immt

inner membrane

1429533_at

protein,MA
NU

cerevisiae)

mitochondrialIdeinsulin degrading
enzyme1423121_at,
1435140_at

keratin 5

1424096_at

Kif1bkinesin family

1423995_at,

ED

Krt5

member 1B

1451642_at,

PT1451200_at,

Elovl6ELOVL family

1425270_at
1417403_atCEmember 6,

elongation of long
chain fatty acids

AC(yeast)Csde1

cold shock domain

1423997_at

containing E1,
RNA binding

Prkca

protein kinase C,

1437393_at,

alpha1446598_at,
1450945_at

Ppm1k

protein

1441988_at

phosphatase 1K(PP2C domain
containing)
Ppp3caSChomolog B (S.

protein

1426401_at,

phosphatase 3,

1438478_a_atcatalytic subunit,

47

ACCEPTED MANUSCRIPT

alpha isoform
Reep1

receptor accessory

1433509_s_at

NADH1426088_at

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P

ND5

T

protein 1

dehydrogenase

Opa1

optic atrophy 1

1434890_at

Slc25a12

solute carrier

1436440_at(mitochondrial
carrier, Aralar),member 12
Slc25a25

solute carrierfamily 25

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family 25

1424735_at,

1447856_x_at(mitochondrial

ED

carrier, phosphate
carrier), member

Tmtc1

PT

25

transmembrane

CE

and

1435261_at

tetratricopeptide
repeat containing 1
transporter 1,

AC

Tap1

1416016_at

ATP-binding
cassette,
sub-family B(MDR/TAP)

Ucp3

uncoupling protein

SCsubunit 51420658_at

3 (mitochondrial,
proton carrier)48