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Supplemental Data Cell Metabolism, Volume 9 Enhanced Orexin Receptor-2 Signaling Prevents Diet-Induced Obesity and Improves Leptin Sensitivity Hiromasa Funato, Allen L. Tsai, Jon T. Willie, Yasushi Kisanuki, S. Clay Williams, Takeshi Sakurai, and Masashi Yanagisawa

Supplemental Figure Legends

Figure S1. Immunohistochemistry for orexin-A in the brain.

(A) Wild-type mouse exhibits orexin-A-positive neurons in the lateral

hypothalamic area (LHA). (B) CAG/orexin mouse shows strong orexin-A

immunoreactivity in the arcuate nucleus (ARH), ventromedial nucleus (VMH),

dorsomedial nucleus (DMH) and LHA, in addition to native orexin-A positive

neurons in LHA. CAG/orexin mouse also shows orexin-A immunoreactivity in the

suprachiasmatic nucleus (SCN)(C), nucleus accumbens shell (AcbSh)(D), globus

pallidus (GP)(E), stratus moleculare of Cornu Ammonis (CA)(F), the hilus of the

dentate gyrus (DG)(F), substantia nigra (SN)(G), area postrema (AP)(H), and

nucleus of the solitary tract (NTS)(H). Abbreviations: aca, anterior branch of the

anterior commissure; AHA, anterior hypothalamic area; CPu, caudate putamen; f,

fornix; LV, lateral ventricle; PVH, paraventricular nucleus; SP, septum; VTA,

ventral tegmental area. Scale bars: 400 μm

The immunohistochemistry for orexin-A was performed as described

previously (Chemelli et al., 1999) with some modifications. In brief, 3-5 month-

old male mice fed a low fat diet were deeply anesthetized with ketamine and

xylazine and transcardially perfused with PBS, and subsequently with 4%

paraformaldehyde. Brains were dissected and postfixed for 12 hr in 4%

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paraformaldehyde before equilibrating in 30% sucrose. Free-floating sections

(35 um-thickness) were incubated in goat-serum before incubated in 1:1000

dilution of rabbit anti-orexin-A antibody (Chemicon, AB3704). Diaminobenzidine

was used to visualize orexin-A immunoreactivity.

Figure S2

Orexin-A-positive neurons and fibers.

(A) Orexin-A-positive neurons and fibers are observed in LHA of wild-type

mouse. (B) CAG/orexin mouse shows similar number of strong orexin-A

immunoreactive neurons and fibers in LHA as wild-type mouse. In addition,

weak immunoreactivities for orexin-A are recognized through LHA. (C) Orexin-A-

positive neurons and fibers are observed in LHA of wild-type mouse with

negligible background staining. (D) In addition to strongly orexin-A-positive

neurons, weak immunoreactivities are recognized in many cell bodies (indicated

by arrowheads). (E) Orexin-A-positive fibers are seen in ARH of wild-type

mouse. (F) In addition to orexin-A-positive fibers, weak orexin immunoreactivity

is recognized through ARH of CAG/orexin mouse. (G) SCN of wild-type mouse

is free from orexin-A-immunoreactivity. (H) CAG/orexin mouse shows weakly to

moderately orexin-A-immunoreactivities in cell bodies in SCN. (I) Dense orexin-

A-positive fibers are seen in dorsal raphe (DR) of wild-type mouse. (J)

CAG/orexin mouse shows dense orexin-A-positive fibers in DR as wild-type

mouse. The scale bars on (A) and (C) indicate 100 μm and 40μm, respectively.

The magnifications of (A) and (C) are same with (B, E, F, I, J) and (D, G, H),

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respectively. (C, D, G, H) are observed using Nomarski differential-interference

contrast.

Figure S3

Immunohistochemistry for orexin-A in the peripheral tissues.

Wild-type mice did not exhibit orexin-A immunoreactivity in the liver (A), adrenal

gland (C), thyroid (E), pancreas (G), skeletal muscle (I), white adipose tissue (K),

and brown adipose tissue (M). CAG/orexin mice exhibited orexin-A

immunoreactivity in the thyroid (F), islet of the pancreas (H) and scattered orexin-

A-positive areas in the medulla of the adrenal gland (D), while no

immunoreactivity was observed in the liver (B), skeletal muscle (J), white adipose

tissue (L), and brown adipose tissue (N). Scale bar: 400 μm

Immunohistochemistry was performed as described in Figure S1 for male mice

fed a low fat diet.

Figure S4

The energy expenditure with effective mass correction and respiratory quotient of

orexin signaling-modified mice on a low fat diet

(A, B) The energy expenditure with effective mass correction (A) and respiratory

quotient (B) over 24 hr of CAG/orexin mice and wild-type mice.

(C, D) The energy expenditure with effective mass correction (A) and respiratory

quotient (B) over 24 hr of OX1R-/-; CAG/orexin mice and OX1R-/- mice.

(E, F) The energy expenditure with effective mass correction (A) and respiratory

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quotient (B) over 24 hr of OX2R-/-; CAG/orexin mice and OX2R-/- mice.

(G, H) The averaged energy expenditure with effective mass correction (G) and

respiratory quotient (H).

Data were sampled every 40 min. Used mice are at the age of 16-20 weeks. The

numbers of mice are 6-9 mice per group. Data are expressed as means ± SEM.

Figure S5

Locomotion of orexin signaling-modified mice.

(A, B) The locomotion over 24 hr of CAG/orexin mice and wild-type mice on a

low fat (A) and a high fat diet (B) .

(C, D) The locomotion over 24 hr of OX1R-/-; CAG/orexin mice and OX1R-/- mice

on a low fat (C) and a high fat diet (D).

(E, F) The locomotion over 24 hr of OX2R-/-; CAG/orexin mice and OX2R-/- mice

on a low fat (E) and a high fat diet (F).

(G) The averaged locomotion of each genotypic group under different fat diet

condition.

Data were sampled every 40 min. Used mice are at the age of 16-20 weeks. The

numbers of mice are 6-9 mice per group. Data are expressed as means ± SEM.

Figure S6

Unaltered preference of CAG/orexin mice over high fat diet and sucrose-

containing water

(A) Both CAG/orexin mice and wild-type mice showed high preference over high

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fat diet. The graph shows the food intake of high fat diet divided by total food

intake for 14 days.

(B) CAG/orexin mice consumed sucrose-containing water during two bottle

choice test in a similar volume as wild-type mice did.

(C) CAG/orexin mice showed preference to sucrose as wild-type mice did.

Two food bottles which contain a low fat diet or a high fat diet were put in

the cage of individually housed male mice. Mice were fed a low fat diet before

food preference test. The food intake for 14 days was measured.

Two drink bottles which contains water or sucrose-containing water were

put in the cage of individually housed mice fed a low fat diet. On the first 3

consecutive days, 3-5 month-old male mice were given the choice of water or 1%

sucrose solution. On next 3 consecutive days, the same mice were given the

choice of water or 10% sucrose solution. The position of two bottles was

switched daily and the daily drink volume was measured. Sucrose preference

was determined by the daily consumption of sucrose-containing water divided by

the daily total fluid consumption. 5-10 mice per group. Data are expressed as

means ± SEM.

Figure S7

Glucose tolerance of CAG/orexin transgenic mice fed a low fat diet.

CAG/orexin transgenic male mice fed a low fat diet has fasted glucose levels

similar to wild-type mice. Likewise, after the administration of glucose, the

glucose levels of CAG/orexin transgenic mice were similar to those of wild-type

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mice at all time points tested.

Glucose tolerance test was performed as described in the method section.

3-4 month-old mice was used. 6-8 mice per group. Data are expressed as

means ± SEM.

Figure S8

Serum TSH, T3, and T4 levels of CAG/orexin mice.

(A) Serum TSH levels of CAG/orexin mice were significantly increased on a

high fat diet compared to those on a low fat diet while there was no significant

difference in serum TSH of wild-type mice on between a low and a high fat diet.

(B, C) Both CAG/orexin mice and wile type mice showed increased levels of T3

and T4 on a high fat condition. There was no significant difference between wilt

type and CAG/orexin mice in the levels of TSH(A) , T3(B), and T4(C).

Blood was collected at the late light phase at the age of 30 weeks. Serum

T3, T4, and TSH was determined by Dr. A. F. Parlow at the National Hormone

and Peptide Program using RIAs. 8-10 mice per group. We used Kruskal-Wallis

test to assess TSH data because there were some samples showing below the

detection limit (48 ng/ml). Data are expressed as means ± SEM.

Figure S9

UCPs mRNA expression in the brown adipose tissue and skeletal muscle.

(A) High fat diet increased UCP1 mRNA of CAG/orexin and wild-type mice.

There was no significant difference in UCP1 mRNA expression between

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CAG/orexin and wild-type mice on each diet condition. Neither UCP2 nor 3

mRNA were affected by diet or by CAG/orexin transgene.

(B) None of UCP1, 2, or 3 mRNA was affected by diet or the CAG/orexin

transgene.

After 2 weeks of high fat diet feeding, we harvested the brown adipose

tissue and skeletal muscle of 3-4 month-old mice. RNA preparation and

quantitative PCR have been done as described in the methods section. UCPs

mRNA expression levels are normalized by GAPDH mRNA level and further

normalized by the average UCPs mRNA expression levels of wild-type mice on

low fat diet. 4-6 mice per group. Data are expressed as means ± SEM.

Figure S10

Adrenalin-related hormone levels and blood pressure of CAG/orexin mice

(A, B) CAG/orexin mice and wild-type mice had comparable levels of

epinephrine (A) and norepinephrine (B) in urine collected for 24 hours.

Urine was collected for 3 consecutive days from male mice fed a low fat

diet. Urinary epinephrine and norepinephrine concentrations were measured

using CatCombi ELISA kit (IBL) and normalized to body weight. Urine was

collected into 0.1N hydrochloric acid solution according to the manufacturer’s

protocol. 5 mice per group.

(C) No significant difference was observed in serum corticosterone levels

between wild-type mice and CAG/orexin mice. Blood was collected at the late

light phase. Serum corticosterone was determined using Corticosterone EIA

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(Immunodiagnostic systems). 8-10 mice per group.

(D) No significant difference was observed in systolic blood pressure between

wild-type mice and CAG/orexin mice under low fat diet or high fat diet. Blood

pressure was measured with tail cuff method using the BP-2000 Blood Pressure

Analysis System II (Visitech Systems) after 4 days of acclimatization. 6 mice per

group. Data are expressed as means ± SEM.

Figure S11

Sleep/Awake analysis during ICV administration of OX2R agonist.

(A) Total time spent in each stage. There was no significant difference in daily

total time spent in wake, NREM, or REM.

(B) Episode duration in each stage. OX2R agonist-infused mice fed a low fat diet

showed longer duration for wake and NREM stages than PBS-infused mice fed a

low fat diet and OX2R agonist-infused mice fed a high fat diet.

We performed surgery for the implantation of an EEG/EMG electrode and

an osmotic pump-connected ICV cannula at a time at day0. Mice were

maintained on a low fat diet before and after surgery. From day9 to day10,

EEG/EMG recording for a low fat diet was performed as described previously

(Chemelli et al. Cell 1999; Willie et al Neuron 2003). Then, the diet was switched

to a high fat diet at day11 and further recorded EEG/EMG until day14. Osmotic

pumps infused OX2R agonist (0.5nmol/day) or PBS in the lateral ventricle for 14

days. 5 mice per group. Statistically significances were shown only within the

same food condition or the same drug infusion condition. Data are expressed as

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means ± SEM.

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Supplementary Figures

Figure S1

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Figure S2

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Figure S3

13

Figure S4

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Figure S5

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Figure S6

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Figure S7

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Figure S8

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Figure S9

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Figure S10

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Figure S11

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Table S1

Immunohistochemistry of Orexin-A

wild type CAG/orexin

Region cell body nerve fiber cell body nerve fiber

Cerebral cortex Cerebral cortex - + - + Anterior olfactory nucleus - ± - ± Olfactory tubercle - ± - ± Piriform cortex - ± - ± Endopiriform nucleus - ± - ± Basal ganglia Accumbens nucleus, shell - ± + + Accumbens nucleus, core - - ± - Lateral septal nucleus - + - + Medial septal nucleus - + - + Nucleus of the diagonal band - + ± + Caudate putamen - - - - Globus pallidus - - - - Bed nucleus of the stria terminalis - ++ ± ++ Anterior cortical amygdaloid nucleus - - - - Postetolateral cortical amygdaloid nucleus

- - - -

Postetomedial cortical amygdaloid nucleus

- - - -

Basolateral amygdaloid nucleus - ± - ± Basomedial amygdaloid nucleus - ± - ± Central amygdaloid nucleus - + - + Lateral amygdaloid nucleus - ± - ± Hippocampal formation

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CA1 - - + - CA2 - - + - CA3 - - + - Dentate gyrus - - + - Subiculum - - - - Thalamus Anteriodorsal thalamic nucleus - - - - Anteriomedial thalamic nucleus - - - - Intermediodorsal thalamic nucleus - ± - ± Mediodorsal thalamic nucleus - - - - Laterodorsal thalamic nucleus - - - - Paraventricular thalamic nucleus - ++ - ++ Reuniens thalamic nucleus - + + + Central medial thalamic nucleus - ++ + ++ Lateral posterior thalamic nucleus - - - - Ventrolateral thalamic nucleus - - - - Ventromedial thalamic nucleus - - - - Reticular thalamic nucleus - - - - Zona incerta - + - + Epithalamus Medial habenular nucleus - - - - Lateral habenular nucleus - + - + Hypothalamus Medial preoptic nucleus - ++ + ++ Paraventricular hypothalamic nucleus - ++ + ++ Suprachiasmatic nucleus - - ++ - Anterior hypothalamic area - ++ + ++ Arcuate hypothalmic nucleus - ++ + ++ Ventromedial hypothalamic nucleus - ++ + ++ Dorsomedial hypothalamic nucleus - ++ + ++

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Lateral hypothalamic area +++ +++ ++++ +++ Posterior hypothalamic area - ++ + ++ Lateral mammillary nucleus - - - - Medial mammillary nucleus - - - - Supramammillary nucleus - ++ - ++ Tuberomammillary nucleus - ++ ± ++ Brain stem and cerebellum Substance nigra, compact part - + + + Substance nigra, reticular part - - - - Ventral tegmental area - ++ + ++ Superior colliculus - ± - ± Inferior colliculus - ± - ± Interpeduncular nucleus - + - + Red nucleus - - - - Cerebellum - - - - Vestibular nucleus - ± - ± Oculomotor nucleus - - - - Motor trigeminal nucleus - - - - Facial nucleus - ± - ± Dorsal motor nucleus of vagus - ± - ± Hypoglossal nucleus - ± - ± Nucleus of the solitary tract - ++ + ++ Area postrema - + + + Parabrachial nucleus - ++ ± ++ Spinal trigeminal nucleus - - - - Dorsal raphe nucleus - ++ ± ++ Median raphe nucleus - ++ ± ++ Dorsal tegmental nucleus - - ± - Laterodorsal tegmental nucleus - ++ + ++ Locus coeruleus - ++ + ++ Periaqueductal gray - ++ ± ++ Pontine nucleus - - - -

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Pontine reticular nucleus - ± - ± Inferior olivary nucleus - - - - Gigantocellular reticular nucleus - + ± +

Except LHA and SCN, orexin-A-immunoreactivity of cell body of CAG/orexin mouse is weak. Nomenclatures were based on Paxinos and Franklin.

Reference Paxinos G, Watson C. The mouse brain in stereotaxic coordinates, 2nd edition, Academic Press. (2000)

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Table S2

Immunohistochemistry of Orexin-A in the peripheral tissues

Wild-type CAG/orexin Brain +++ +++ Liver - -

Kidney - - Thyroid - ++ Adrenal - +

Pancreas (Exocrine) - - Pancreatic islet - ++ Skeletal muscle - - White fat tissue - - Brown fat tissue - -

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Table S3

Distribution of orexin receptors in peripheral tissues

Tissue Method OX1R OX2R Species Reference

Northern − − Rat Sakurai (1998) Heart RT-PCR − − Rat Johren (2001) RT-PCR, IHC

+ − Rat Kirchgessner (1999)

Stomach

RT-PCR − − Rat Johren (2001) Jejunum qPCR − − Mouse This study

Northern − − Rat Sakurai (1998) RT-PCR − − Rat Johren (2001)

Liver

qPCR − − Mouse This study RT-PCR − − Rat Johren (2001) RT-PCR + + Rat Kirchgessner

(1999) RT-PCR + + Rat Nowak (2005)

Pancreas

qPCR − − Mouse This study IHC + − Rat Kirchgessner

(1999) Pancreatic islet

qPCR 3% of brain expression level

30% of brain expression level

Mouse This study

RT-PCR + ND Rat Johren (2001) Thyroid gland qPCR 3% of brain

expression level

2% of brain expression level

Mouse This study

RT-PCR, IHC

− + Human Randeva (2001)

IHC + − Human Blanco (2002) RT-PCR, IHC

+ + Human, rat

Spinazzi (2005)

Adrenal cortex

RT-PCR − + Rat Johren (2001)

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RT-PCR + + Rat Lopez (1999) RT-PCR − − Rat Johren (2001)

Adrenal medulla

IHC − + Human Blanco (2002) RT-PCR − − Rat Johren (2001) RT-PCR, IHC

+ + Human Digby (2006) White adipose tissue

qPCR − − Mouse This study Northern − − Rat Sakurai (1998) Skeletal

muscle RT-PCR − − Rat Johren (2001)

IHC, immunohistochemistry; RT-PCR, reverse transcriptase PCR; qPCR, quantitative

RT-PCR; +, detected; −, not detected; ND, not done.

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