Page 1 of 42 Diabetes

Hypothalamic orexin prevents hepatic insulin resistance via daily

bidirectional regulation of autonomic nervous system in mice

Hiroshi Tsuneki,1 Emi Tokai,1 Yuya Nakamura,1 Keisuke Takahashi,1 Mikio Fujita,1

Takehiro Asaoka,1 Kanta Kon,1 Yuuki Anzawa,1 Tsutomu Wada,1 Ichiro Takasaki,2

Kumi Kimura,3 Hiroshi Inoue,3 Masashi Yanagisawa,4,5 Takeshi Sakurai,6 and Toshiyasu

Sasaoka1

1Department of Clinical Pharmacology, 2Division of Molecular Genetics Research, University

of Toyama, 2630 Sugitani, Toyama 9300194, Japan.

3Frontier Science Organization, Kanazawa University, Kanazawa, Ishikawa 9208641, Japan.

4Howard Hughes Medical Institute, Department of Molecular Genetics, University of Texas

Southwestern Medical Center, Dallas, Texas 75390, U.S.A.

5Center for Behavioral Molecular Genetics, University of Tsukuba, Tsukuba, Ibaraki 305

8575, Japan.

6Department of Molecular Neuroscience and Integrative Physiology, Faculty of Medicine,

Kanazawa University, Kanazawa, Ishikawa 9208640, Japan.

Short running title: Regulation of metabolic rhythm by orexin

Corresponding Author: Hiroshi Tsuneki, Ph.D. or Toshiyasu Sasaoka, M.D., Ph.D.

Tel.: +81764347550, Fax: +81764345067, Email: [email protected]toyama.ac.jp or

[email protected]toyama.ac.jp

H.T. and E.T. contributed equally to this study.

Number of Figures: 6

Supporting documents/data: 6

1 Diabetes Publish Ahead of Print, published online September 23, 2014 Diabetes Page 2 of 42

Abstract

Circadian rhythm is crucial for preventing hepatic insulin resistance, although the mechanism remains uncovered. Here we report that wakeactive hypothalamic orexin system plays a key role in this regulation. Wildtype mice showed a daily rhythm in blood glucose levels peaked at awake period; however, the glucose rhythm was disappeared in orexin knockout mice despite normal feeding rhythm. Central administration of orexin A during nighttime awake period acutely elevated blood glucose levels but subsequently lowered daytime glucose levels in normal and diabetic db/db mice. The glucoseelevating and lowering effects of orexin A were suppressed by adrenergic antagonists and hepatic parasympathectomy, respectively.

Moreover, the expression levels of hepatic gluconeogenic , including Pepck, were increased and decreased by orexin A at nanomolar and femtomolar doses, respectively. These results indicate that orexin can bidirectionally regulate hepatic via control of autonomic balance, leading to generation of the daily blood glucose oscillation. Furthermore, during aging, orexin deficiency enhanced endoplasmic reticulum (ER) stress in the liver, and caused impairment of hepatic insulin signaling and abnormal gluconeogenic activity in pyruvate tolerance test. Collectively, the daily glucose rhythm under control of orexin appears to be important for maintaining ER homeostasis, thereby preventing insulin resistance in the liver.

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INTRODUCTION

Maintaining glucose and energy homeostasis throughout the day is essential for survival.

Therefore, different metabolic functions are timely activated according to the circadian

rhythm. Recently, it has also become clear that fine tuning of daily metabolic rhythms is

crucial for preventing metabolic abnormalities. For instance, chronic disruption of circadian

rhythm in , as seen in shift workers, increases the risk of obesity and type 2 diabetes

(1,2), and circadian rhythms of glucose regulation are impaired in diabetic animals and

patients with diabetes (3).

Circadian rhythms of peripheral tissues, including liver, are entrained to the central

biological located in the suprachiasmatic nuclei (SCN) via autonomic and hormonal

pathways (4). The liver plays a pivotal role in maintaining optimal glucose levels via hepatic

glucose production (HGP) and glucose uptake. The daily rhythm of HGP is regulated by

autonomic nervous system under SCN control, leading to generation of a daily rhythm in

basal blood glucose levels independently of the feeding rhythm (5). The central clock is also

crucial for preventing metabolic abnormalities in the liver, since lesions of the SCN caused

severe hepatic insulin resistance in mice (6); however, the underlying mechanisms remain

uncertain. It is of note that nutrient excess causes endoplasmic reticulum (ER) stress in

hepatocytes, a main cause of hepatic insulin resistance, and the cells need “resting time” to

recover from the ER stress (7,8). These raise a possibility that daily changes in blood glucose

levels are required for maintaining ER homeostasis and insulin sensitivity in the liver.

The perifornical hypothalamic area is one of the primary sites for mediating the functional

interplay between the brain and liver (9). Orexin A and B, a pair of neuropeptides, are

produced in neurons in the perifornical and lateral hypothalamus. Importantly, the SCN sends

signals to orexinproducing neurons via the dorsomedial hypothalamus (10,11), and controls

the orexin secretion (12). The central orexin A concentrations exhibit a daily rhythm with the

3 Diabetes Page 4 of 42

highest levels during wakefulness (13,14), although the orexin expression was down regulated in diabetic db/db and ob/ob mice (15). Orexin regulates the sleepwake rhythm, energy homeostasis, and autonomic nerve balance through orexin 1 (OX1R) or 2

(OX2R) (16). Moreover, orexin appears to be involved in the maintenance of insulin sensitivity, because orexindeficient male mice fed normal chow diet exhibited agerelated development of systemic insulin resistance despite normal body weight (17,18), and because orexindeficient narcoleptic patients with cataplexy showed body mass indexindependent metabolic alterations, including insulin resistance (19). However, the role of orexin in glucose homeostasis remains elusive, since orexin A is reported to cause the blood glucoseelevating and lowering effects in rodents depending on the experimental conditions (2022). We hypothesized that orexin has the ability to promote generation of the glucose rhythm via bidirectional regulation of HGP. Moreover, the orexincontrolled glucose rhythm, if any, would help to prevent hepatic ER stress and insulin resistance.

The aim of the present study was therefore to clarify, for the first time, whether hypothalamic orexin acts as a timekeeper in daily glucose regulation, and whether it contributes to the maintenance of hepatic insulin sensitivity. To this end, we examined the influence of orexin deficiency and orexin A administration on daily changes in blood glucose levels and the regulation of hepatic gluconeogenesis in mice. Furthermore, we investigated whether orexin deficiency causes excessive ER stress in the liver, leading to hepatic insulin resistance during aging.

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RESEARCH DESIGN AND METHODS

Animals

Mice were housed at 2325°C with free access to normal chow diet and water under a 12:12 h

lightdark cycle (lights on from 0700 to 1900 h). Zeitgeber time (ZT) 0 and 12 are defined as

lightson and off times, respectively. The animal experiments were performed between 1000

and 1600 h, unless otherwise indicated. All experimental procedures used in this study were

approved by the Committee of Animal Experiments at the University of Toyama or Kanazawa

University. Male C57BL/6J mice, male C57BLKS/J Iarm+/m+ mice (m+/m+) and male

C57BLKS/J Iar+Leprdb/+Leprdb mice (db/db) were purchased from Japan SLC (Shizuoka,

Japan). Wildtype (WT) mice, preproorexindeficient (Orexin/) mice, Ox1rdeficient (Ox1r/

) mice, and Ox2r−/− mice (N5N6 backcross to C57BL/6J) were prepared as described

previously (17,23). Continuous measurements of the amounts of food intake, energy

consumption, and locomotor activity were performed using metabolic cages (MK5000RQ;

Muromachi Kikai, Tokyo, Japan).

Materials

Orexin A was purchased from the Peptide Institute (Osaka, Japan). regular insulin

Novolin R was provided by Novo Nordisk (Copenhagen, Denmark). Antiinsulin receptor

substrate 1 (IRS1) and antiIRS2 antibody were purchased from Millipore

(Temecula, CA), and an antiphosphoinositolrequiring 1α (IRE1α) (Ser724) antibody

was from Abcam (Cambridge, MA). Antiinsulin receptor β antibody and antiAkt1 antibody

were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). AntiIRE1α antibody, anti

phosphoSAPK/Jun Nterminal kinase (JNK) (Thr183/Tyr185) antibody, antiphosphoAkt

(Ser473) antibody, antiβ antibody, anticyclic AMP response element binding

(CREB) antibody, antiphosphoCREB (Ser133) antibody, antimammalian target of

rapamycin (mTOR) antibody, antiphosphomTOR (Ser2448) antibody, antisignal transducer

5 Diabetes Page 6 of 42

and activator of 3 (STAT3) antibody, and antiphosphoStat3 (Tyr705) antibody were from Cell Signaling Technology (Beverly, MA).

Biochemical analyses of blood and tissue samples

Blood samples were collected from the tail vein or orbital sinus of mice under anesthesia.

Serum levels of insulin (Takara Bio, Shiga, Japan), glucagon (Wako Pure Chemical, Osaka,

Japan), and corticosterone (Cayman Chemical Company, Arbor, MI) were measured with specific ELISA. Fasting serum levels of triglyceride, nonesterified fatty acids (NEFA), and cholesterol were determined using colorimetric kits (Wako). Blood glucose levels were measured using a glucose meter (FreeStyle Freedom, Nipro, Osaka, Japan). Tissue contents of glycogen were measured using a glucose assay kit (SigmaAldrich).

Intracerebroventricular (ICV) administration

ICV administration of orexin A (ICVorexin A) was performed as described previously (17).

In brief, a guide cannula was implanted into the lateral ventricle of mice. After 7day recovery period, the mice were ICV injected with 3 l saline or orexin A using a Hamilton syringe. For the glucose clamp study, as described below, an ICV injection cannula with extension tubing (Plastics One, Roanoke, VA) was used for the drug injection. Hepatic parasympathectomy of C57BL/6J mice at 7 weeks old was performed immediately before the

ICV cannulation using standard protocols as described previously (24). Furthermore, orexin

A was administered continuously according to a previously described method (25). In brief, an osmotic pump (Alzet 1007D; Durect, Cupertino, CA) containing saline was subcutaneously placed on the back, and an ICV cannula connected to the pump (Alzet brain infusion kit 3; Durect) was implanted into the lateral ventricle in 7weekold db/db mice under anesthesia. After a 7day recovery period, the mice were administered either orexin A

(0.3 nmol/day) or saline by replacing the pump containing each solution.

Physiological analyses of hepatic glucose production

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Hyperinsulinemiceuglycemic clamp studies were performed as described previously with

minor modification (26). After 7 days of recovery from ICV cannulation surgery, 8weekold

C57BL/6J mice were fasted for 16 h, and injected with orexin A or saline through the ICV

cannula. [33H]Glucose infusion at 0.1 Ci/min was started 60 min after the ICV injection.

For measurement of the basal HGP rate, blood samples were collected 120 min after the ICV

injection. Subsequently, insulin was infused at 1.25 mU/kg/min with variable amounts of

40% glucose solution to maintain a blood glucose level of 90120 mg/dL. The glucose

infusion rate was measured between 90 and 120 min after the initiation of insulin infusion to

calculate the HGP rate. For the pyruvate tolerance test to evaluate the activity of HGP, fasted

mice were intraperitoneally injected with pyruvate (2 g/kg), and blood samples were collected

from the tail vein.

Western blot analysis

The tissue samples dissected from mice were homogenized, lysed and subjected to

immunoblotting, as previously described (17). For the analysis of insulin signaling, mice

fasted overnight were injected with insulin or PBS. Fifteen minutes after injection, tissues

were rapidly isolated.

Reverse transcription-PCR.

RNA extraction, reverse transcription, and quantitative realtime PCR were performed using a

Mx3000p realtime PCR system (Stratagene, La Jolla, CA) with SYBR Premix Ex Taq II

(Takara Bio), as described previously (25). Expression level of the transcript of interest

was calculated as a ratio to that of S18 ribosomal protein in each sample. Primer pairs are

listed in Supplementary Table 1.

Microarray and pathway analyses

The liver tissues were isolated at ZT 8 from male WT and Orexin/ mice at 6 months old

under fed conditions. Total RNA pooled from two mice was used to synthesize doublestrand

7 Diabetes Page 8 of 42

cDNA for each experimental group. The gene transcript profiles were analyzed using a

GeneChip system with Mouse Genome 430 2.0 Array (Affymetrix, Santa Clara, CA),

GeneSpring software (Agilent Technologies, Santa Clara, CA), and Ingenuity Pathway

Analysis software (Ingenuity Systems, Mountain View, CA), as described previously (27).

Electron microscopy

The liver tissues isolated from mice were immediately fixed with 2% glutaraldehyde in 0.1 M

PBS (pH 7.4) for 90 min at 4 °C, and postfixed with 1% osmium tetroxide for 90 min at 4 °C.

The specimen was dehydrated, embedded in Epoxy resin and sectioned using standard protocols. Ultrathin sections (70 nm thickness) were stained with uranyl acetate and lead citrate. Electron micrographs were taken with a transmission electron microscope (JEOL

JEM1400TC, Tokyo, Japan).

Statistical analysis

Data are expressed as the means ± S.E.M. The significance of differences between two groups was assessed by Student’s t test, and differences between multiple groups were assessed by oneway ANOVA followed by the Bonferroni test. Daily rhythm in blood glucose levels was determined using Time Series Analysis Serial Cosinor 6.3 software (Expert Soft Technologie,

France). P<0.05 was considered significant.

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RESULTS

Orexin dually regulates HGP and generates daily blood glucose rhythm in mice

Blood glucose levels exhibited daily changes that peaked at ZT 14 in the dark phase in 3 and

6monthold WT mice under ad libitum fed conditions; however, Orexin/ mice at 3 and 6

months old did not show the increase in blood glucose at ZT 14, when compared to ZT 5 (Fig.

1A and B). Cosinor analysis showed the presence of a significant daily rhythm in WT mice at

both age and 3monthold Orexin/ mice (P<0.01) but not in 6monthold Orexin/ mice

(P=0.725). In addition, the glucose levels from ZT 5 to 11 and ZT 20 to 2 were significantly

higher in 6monthold Orexin/ mice than those in WT mice (Fig. 1B). The timing of feeding

and locomotion in Orexin/ mice was identical to that of WT mice at both age, although their

absolute activities in the dark phase were lower in Orexin/ mice (Fig. 1CF). Body weights

and 24h energy expenditure were not different between the two groups (data not shown).

Serum levels of insulin and glucagon in Orexin/ mice under fed condition were almost

comparable with those of WT mice: the detected changes in the hormone levels were not

reflected in the glucose changes (Fig. 1GJ). Serum levels of corticosterone, triglyceride,

NEFA, and total cholesterol were not different between WT and Orexin/ mice (data not

shown). Thus, orexin deficiency preferentially affected daily changes in glucose levels.

Since daily glucose rhythm largely depends on HGP, profiles of hepatic gluconeogenic

in Orexin/ mice were investigated. At 3 months of age, Orexin/ mice

exhibited decrease in the mRNA levels of peroxisome proliferatoractivated receptor γ

1α (Pgc1a) at ZT14 (Fig. 1K). The expression of glucose6phosphatase (G6Pase)

was also deranged in the light phase (Fig. 1L), although the Pepck expression was unaltered

(data not shown). At 6 months of age, Orexin/ mice exhibited significant increase in the

expression of Pepck at ZT8 and a trend of increase in the expression of G6Pase (Fig. 1M and

9 Diabetes Page 10 of 42

N) and Pgc1a (data not shown) at ZT 8. Thus, orexin deficiency perturbed the daily rhythms of hepatic gluconeogenic gene expression.

To characterize the effect of orexin A on blood glucose levels, orexin A at 3 nmol was

ICV injected into C57BL/6J mice. After the injection, blood glucose increased and peaked at

60 min (Fig. 2A). This effect was inhibited by combined pretreatment of α and βadrenergic antagonists (phenoxybenzamine and propranolol) (Fig. 2B). ICVorexin A at 3 nmol caused an increase in the basal rate of HGP assessed by a tritiated glucose infusion method in

C57BL/6J mice, although orexin A had no effect on HGP during hyperinsulinemic euglycemic clamp (Fig. 2C).

Diabetic db/db mice exhibited hyperglycemia throughout the day, although there existed a significant daily rhythm in blood glucose (P<0.01, determined by Cosinor analysis) peaked at

ZT 17 (Supplementary Fig. 1A). In addition, preproorexin mRNA levels in the hypothalamus in db/db mice were lower than those in C57BL/6J mice (Supplementary Fig. 1B). We therefore examined the influence of nighttime orexin A supplementation on blood glucose. In db/db mice fasted for 6 h, ICVorexin A at 0.3 nmol did not significantly affect the glucose levels (Fig. 2D), whereas ICVorexin A at 3 nmol acutely increased blood glucose (Fig. 2E).

In nondiabetic m+/m+ mice, both doses of orexin A significantly elevated the glucose levels

(Fig. 2D and 2E). To further reveal the impact of orexin A on daily glucose rhythm, orexin A was administered daily to db/db mice at ZT 14 under ad libitum fed condition, since cerebrospinal fluid levels of orexin A increase in the dark phase in rodents (14). Intriguingly, the first nighttime administration of orexin A at 0.3 nmol had no acute effect on blood glucose, but gradually lowered it in the subsequent light phase (Fig. 2F). Moreover, the second and third administrations caused the rapid glucose elevation at ZT 15 to 16, followed by the lowering of glucose at ZT 2, thereby generating an apparent glucose oscillation. Similarly, in

20weekold db/db mice, ICVorexin A at 0.3 nmol reduced fed glucose levels at ZT 14 (Fig.

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2G) without affecting body weight (Fig. 2H). It also reduced fasting glucose and hepatic

Pgc1α, Pepck and G6Pase without affecting serum insulin at ZT 8 (Fig. 2IK). Under these

conditions, increased phosphorylations of CREB, JNK, and mTOR in the liver of db/db mice,

relevant to hyperglycemia, were reduced to nondiabetic control levels (Supplementary Fig.

2AC). ICVorexin A at ZT 14 for 3 days had no effect on insulininduced Akt

phosphorylation in the of db/db mice dissected at ZT 6 (Supplementary Fig.

3A). Daytime administration (at ZT 2) of orexin A at 0.3 nmol was obviously less effective at

lowering glucose in db/db mice (Fig. 2L) than its nighttime administration (at ZT14) as shown

above (Fig. 2F). In addition, continuous ICV infusion of orexin A (0.3 nmol/day) using an

osmotic pump did not affect blood glucose levels and body weight in db/db mice (Fig. 2M),

whereas hepatic G6Pase was increased by the orexin infusion (Fig. 2N). These results indicate

that daily increase in central orexin levels in the dark phase contributes to the onset of glucose

oscillation via adrenergic pathway.

To investigate whether adrenaline substitutes for orexin to induce daily glucose oscillation,

adrenaline was injected intraperitoneally into db/db mice at ZT14 under fed conditions.

Adrenaline acutely caused shortterm elevation of blood glucose; however, it failed to affect

glucose levels in the light phase (Fig. 3A). Thus, the glucose elevation by sympathetic signals

was not enough for subsequent lowering of blood glucose. After ICV administration of orexin

A at 3 nmol, hepatic STAT3 phosphorylation, indicative of parasympathetic input to the liver

(28), was increased at 120 min in db/db mice compared to respective saline controls

(Supplementary Fig. 2D). We therefore conducted hepatic parasympathectomy experiments to

investigate the involvement of parasympathetic pathway in the glucoselowering effect of

orexin. In shamoperated C57BL/6J mice, ICVorexin A (3 nmol) at ZT 14 enhanced the

daily changes in fed glucose levels (Fig. 3B): the rapid glucose elevations in the dark phase by

the second and third administrations were followed by lowering of glucose in the subsequent

11 Diabetes Page 12 of 42

light phase, when compared to the saline control. No change was observed in body weight and food intake by daily orexin A administration (data not shown). After the orexin treatment, blood glucose levels at 15 min in pyruvate tolerance test were slightly decreased compared to the saline control (Fig. 3C). In contrast, hepatic parasympathectomy abolished the glucose lowering effect of orexin A in the light phase (Fig. 3D), whereas acute increases in glucose levels by orexin A in the dark phase were not statistically different between shamoperated and parasympathectomized mice (Fig. 3B and D). Moreover, in the subsequent pyruvate tolerance test, the glucose elevation at 15 min was greater in the orexintreated group than the saline control group (Fig. 3E). Therefore, hepatic parasympathetic activity appears to be required for the glucoselowering effect of orexin A. Neither ICVorexin A at ZT 14 for 3 days nor hepatic parasympathectomy affected insulininduced Akt phosphorylation in the skeletal muscle at ZT 6 (Supplementary Fig. 3B).

To further reveal the mode of action of orexin A on hepatic glucose regulation, the dose response relationship was examined. Under fasting condition, blood glucose levels were elevated by ICVorexin A at 3 nmol, but lowered at 3 fmol (Fig. 4A). Serum levels of insulin were not altered by these treatments (Fig. 4B). Consistently, the expression levels of hepatic gluconeogenic genes, Pepck and G6Pase, were increased by ICVorexin A at 3 nmol, but decreased at 3 fmol (Fig. 4C and D). In the liver of Ox1r/ mice, the decreasing effect of ICV orexin A at 3 fmol on the Pepck mRNA levels was disappeared, whereas ICVorexin A at 3 nmol normally increased the Pepck levels (Fig. 4E). In contrast, ICVorexin A at both 3 fmol and 3 nmol decreased the hepatic Pepck levels in Ox2r/ mice (Fig. 4F). The orexin A (3 nmol)induced increase in Pepck expression was inhibited by pretreatment with an adrenergic

αblocker phenoxybenzamine (Fig. 4G) but not a muscarinic antagonist atropine (data not shown), whereas the orexin A (3 fmol)induced decrease in Pepck expression was inhibited by pretreatment with atropine (Fig. 4H) but not phenoxybenzamine (data not shown).

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Therefore, it is likely that the highdose of orexin A enhances the Pepck levels via the OX2R

and sympathetic nerve pathways, whereas the lowdose of orexin A suppresses them via the

OX1R and parasympathetic pathways.

Orexin-induced daily glucose rhythm prevents ER stress and hepatic insulin resistance

Next we investigated the impact of longterm deficiency of the daily orexin action on

hepatic glucose . First, the expression profiles of core clock components in the

liver were compared between WT and Orexin/ mice, since the biological clock system serves

to control metabolic efficacy (2). The mRNA levels of Period 2 (Per2) at ZT 8 were

significantly, and those of Per1 were slightly, increased in 6monthold Orexin/ mice,

whereas no changes were detected at 3 months old (Supplementary Fig. 4). Moreover, since

the glucose levels and hepatic Pepck levels were deranged mainly during daytime in Orexin/

mice along with aging (Fig. 1), we explored the underlying mechanism in the light phase. In

the pyruvate tolerance test, the glucose response was greater in both male and female Orexin/

mice at 6 months old (Fig. 5A and B) but not 3 months old (data not shown), when compared

to agematched WT controls. Glycogen contents in the liver and skeletal muscle were not

different between WT and Orexin/ mice at 6 months old (data not shown). Concerning the

insulin signaling, phosphorylation of Akt at Ser473 induced by insulin was significantly

reduced in the liver but not skeletal muscle of Orexin/ mice at 6 months old compared to WT

controls (Fig. 5C). The mRNA levels of Irs2 and the protein levels of IRS1 and IRS2 (Fig. 5D

and E), but not insulin receptor (not shown), were significantly decreased in the liver of 6

monthold Orexin/ mice under fed conditions. The GeneChip and pathway analyses indicated

that the gene expression profile in the liver of 6monthold Orexin/ mice at ZT 8 under fed

conditions was highly related to diabetes mellitus and glucose metabolism disorder

(Supplementary Table 2). On the other hand, the mRNA levels of tumor necrosis factor α

13 Diabetes Page 14 of 42

(Tnfa) and interleukin1β (Il1b) in epididymal fat tissues were not different between WT and

Orexin/ mice (data not shown). Thus, chronic deficiency of daily orexin action perturbs the clock system in the liver and impairs the regulation of HGP in the light phase.

ER is an organelle functionally linked to the circadian clock and insulin sensitivity (7,29).

Under 5h fasting conditions, the mRNA levels of C/EBPhomologous protein (Chop), an ER stress marker, were increased in the liver of Orexin/ mice at 6 months old (Fig. 6A) but not at

3 months old (data not shown). When ER stress was induced by 1h refeeding after the 24h fasting period, according to a previously reported ERstress paradigm (30), abnormal increases in the phosphorylation of IRE1α (Fig. 6B) and JNK (Fig. 6C) were observed in the liver of Orexin/ mice at 6 months old compared to those of WT controls. Similarly, excessive phosphorylation of JNK was induced by refeeding after fasting for 24 h in the liver of Ox1r/ and Ox2r/ mice at 4 months old compared to those of WT mice (Fig. 6D). Electron microscope analysis demonstrated that Orexin/ mice at 6 months old displayed typical features of ER stress, i.e. the expansion and dilation of ER in the liver (Fig. 6E). Thus, lack of daily orexin action caused agerelated development of ER stress in the liver, relevant to hepatic insulin resistance.

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DISCUSSION

The SCNcontrolled biological clock rhythm is crucial for maintaining hepatic insulin

sensitivity (6). In the present study, we investigated the underlying mechanisms, and found

that 1) hypothalamic orexin bidirectionally regulates hepatic gluconeogenesis via control of

autonomic balance, leading to generation of the daily blood glucose oscillation. We further

found that 2) the orexininduced periodical activation and inactivation of hepatic

gluconeogenesis reduces ER stress in the liver, thereby preventing hepatic insulin resistance.

These results provide the first evidence that orexin plays a key role in the maintenance of

hepatic insulin sensitivity by circadian system.

The SCN controls daily glucose rhythm independently of feeding rhythm. The daily

glucose oscillation peaks at the time of awakening mainly through increase in HGP (9,31).

The balance between sympathetic and parasympathetic output to the liver is crucial for

maintenance of the glucose rhythm (32), although the integrating mechanism has long been

uncertain. We found that central action of orexin A at awake period increased the amplitude

of daily glucose oscillations via HGP in mice. Moreover, deficiency of endogenous orexin

disrupted the daily glucose rhythm. These suggest that orexin mediates the regulation of HGP

rhythm by circadian system.

Hypothalamic orexin neurons project to both sympathetic and parasympathetic

preganglionic neurons in the central nervous system (33,34), and orexin activates both type of

neurons (35,36). In fact, renal sympathetic nerve activity and blood pressure were increased

by ICVorexin A at high doses (3 pmol to 3 nmol) but decreased at low dose (3 fmol) in rats

(37). Consistently, we found that ICVorexin A at 3 nmol enhanced hepatic gluconeogenic

gene expression via αadrenergic pathway, whereas ICVorexin A at 3 fmol suppressed it via

cholinergic pathway in fasted mice. These raise a possibility that when local concentrations of

orexin A increase and then decrease after ICV administration, the mode of action may be

15 Diabetes Page 16 of 42

converted from sympathetic to parasympathetic nervemediated one. Indeed, high dose (3 nmol) of orexin A at awake period caused sympathetic pathwaydependent blood glucose elevation that was accompanied by parasympathetic pathwaydependent lowering of glucose at resting period. Therefore, daily changes in the orexinergic tones under control of circadian system appear to serve as a master ‘onoff’ signal for HGP.

In diabetic mice such as db/db and ob/ob mice, orexin expression is suppressed due to hyperglycemia (15, 38). We found that ICVorexin A more profoundly reduced blood glucose levels in db/db than C57BL/6J mice. These suggest that physiological orexin A action plays a fundamental role in the maintenance of glucose homeostasis, and supplementation of orexin A to ameliorate its suppressed level in diabetic state can improve hyperglycemia. In addition, unlike normal daily glucose oscillation peaked at ZT 14, the highest glucose levels were observed at ZT 17 in db/db and 6monthold Orexin/ mice, implying that the peaktime change in db/db mice may be related, at least in part, to orexin insufficiency. Importantly, the present chronopharmacological approach demonstrated that the timing of orexin A administration is crucial for glucose regulation: its administration at sympatheticdominant nighttime awake period, that mimics the physiological orexin secretion pattern, was more effective to lower blood glucose than parasympatheticdominant daytime resting period. The diabetic db/db mice have been reported to exhibit the increased sympathetic and decreased parasympathetic tones in autonomic cardiovascular regulation (39). Increased sympathetic tone enhances parasympathetic control, leading to an accentuated antagonism in heart (40).

By similar mechanism, the parasympathetic control of hepatic glucose production by orexin may be enhanced, when the sympathetic tone is increased in the diabetic state and/or during the active phase.

Interestingly, ICVorexin A at both high and low doses exclusively suppressed hepatic

Pepck expression in Ox2r/ mice, whereas they only increased it in Ox1r/ mice. Therefore,

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hepatic gluconeogenesis may be enhanced by OX2R/sympathetic signals and suppressed by

OX1R/parasympathetic signals (Fig. 6F). However, since OX1R and OX2R are widely and

differentially distributed throughout the brain (41), more precise study is required to clarify

the contribution of each receptor subtype in various brain regions to hepatic glucose

regulation.

In type 2 diabetic subjects, fasting plasma glucose levels were elevated due to the increase

in basal HGP which is entirely explained by an increase in hepatic gluconeogenesis (42). In

our study, Orexin/ mice at 6 months old exhibited hyperglycemia associated with increased

activity of hepatic gluconeogenesis at daytime resting period, despite normal feeding behavior.

These abnormalities appear to be due to hepatic insulin resistance, since 6monthold Orexin/

mice exhibited impairment of insulin signaling in the liver but not skeletal muscle. Systemic

insulin resistance, glucose intolerance, and impaired insulin signaling in the skeletal muscle

become apparent in Orexin/ mice older than 9 months old, as reported previously (17). Thus,

orexin deficiency appears to cause insulin resistance preferentially in the liver during the

aging process.

The ER stress response is a shortterm adaptive system to cope with abnormal increase in

unfolded in the ER lumen; in the liver, protein synthesis for metabolic

reprogramming is promoted during postprandial state or by refeeding after fasting, resulting in

the development of ER stress (43). However, prolonged ER stress is detrimental and leads to

the development of insulin resistance and type 2 diabetes (44,45). In this deleterious process,

the IRE1α/JNK signaling pathway plays a major role. In our study, abnormal increase in

hepatic IRE1α/JNK signaling was induced by refeeding after 24h fasting in the liver of

Orexin/ mice at 6 months but not 3 months old. Since the glucose rhythm was already

disrupted in 3monthold Orexin/ mice, nonrhythmic HGP throughout the day may

gradually impair the machinery for protecting ER stress in the liver. Ox1r/ and Ox2r/ mice

17 Diabetes Page 18 of 42

exhibited similar abnormalities, suggesting that the absence of daily action of orexin, rather than the complete lack of orexin, is the main cause of the increased ER stress. Periodical action of endogenous orexin under the SCN control may provide the time for recovery from

ER stress in the liver by suppressing HGP at resting period, and prevent hepatic insulin resistance (Fig. 6F).

Although glucose variability is supposed to be a causal factor in the development of vascular complications or mortality, a larger study did not find any relation between them; therefore, so far, there is little consensus on the importance of glucose variability (46). Rather, it has been reported that nighttimerestricted feeding accompanied by nocturnal increase in carbohydrate utilization improved metabolic state in mice fed highfat diet (47). Moreover,

ER stress can be mitigated by lowlevel preconditioning stimuli (48), and circadian clock dependent rhythmic activation of the IRE1α signaling in the ER of liver is required for normal metabolic functions in mice (29). Therefore, daily oscillation of basal glucose levels under orexin control is considered to be beneficial for preventing metabolic disorders.

In conclusion, the present study indicated that orexin functions as a timekeeper to regulate the daily onoff rhythm in HGP along the sleepwake cycle. Importantly, this mechanism was required for not only supplying energy at the time of awakening but also preventing the development of hepatic insulin resistance (Fig. 6F). Therefore, the present results may provide a basis for the development of novel chronotherapy against metabolic diseases associated with circadian rhythm disturbance, including type 2 diabetes. Pharmacological or nonpharmacological intervention to augment the amplitude of daily change in orexinergic tone might be therapeutically valuable.

18 Page 19 of 42 Diabetes

Acknowledgments. The authors thank K. Yamaguchi, C. Sugawara, T. Nagata, K. Nishio,

and Dr. H. To (University of Toyama, Toyama, Japan) for technical assistance.

Funding. This work was supported by JSPS KAKENHI Grant Number 24591317,

24390056, and grants from the Takeda Science Foundation (H.T.), the Smoking Research

Foundation (H.T.), the Research Foundation for Pharmaceutical Sciences (H.T.), the Japan

Diabetes Foundation (T.S.), the Foundation for Growth Science (T.S.), and the Tamura

Science and Technology Foundation (T.S.).

Duality of Interest. No potential conflicts of interest relevant to this article were reported.

Author Contributions. H.T. researched the data and wrote the manuscript. E.T., Y.N.,

K.T., M.F., T.A., K.K., Y.A., I.T., K.K., and H.I. researched the data. T.W., M.Y., T.S., and

T.S. contributed to discussion and wrote the manuscript. H.T. and T.S. are the guarantors of

this work and, as such, had full access to all the data in the study and take responsibility for

the integrity of the data and the accuracy of the data analysis.

19 Diabetes Page 20 of 42

References

1. Froy O. Metabolism and circadian rhythms: implications for obesity. Endocr Rev

2010;31:124

2. Sahar S, SassoneCorsi P. Regulation of metabolism: the circadian clock dictates the time.

Trends Endocrinol Metab 2012;23:18

3. Huang W, Ramsey KM, Marcheva B, Bass J. Circadian rhythms, sleep, and metabolism. J

Clin Invest 2011;121:21332141

4. Kalsbeek A, Yi CX, Cailotto C, la Fleur SE, Fliers E, Buijs RM. Mammalian clock output

mechanisms. Essays Biochem 2011;49:137151

5. Kalsbeek A, Bruinstroop E, Yi CX, Klieverik LP, La Fleur SE, Fliers E. Hypothalamic

control of energy metabolism via the autonomic nervous system. Ann NY Acad Sci

2010;1212:114129

6. Coomans CP, van den Berg SA, Lucassen EA, et al. The suprachiasmatic nucleus controls

circadian energy metabolism and hepatic insulin sensitivity. Diabetes 2013;62:11021108

7. Eizirik DL, Cardozo AK, Cnop M. The role for endoplasmic reticulum stress in diabetes

mellitus. Endocr Rev 2008;29,4261

8. Cnop M, Foufelle F, Velloso LA. Endoplasmic reticulum stress, obesity and diabetes.

Trends Mol Med 2012;18,5968

9. Kalsbeek A, Yi CX, La Fleur SE, Fliers E. The hypothalamic clock and its control of

glucose homeostasis. Trends Endocrinol Metab 2010;21,402410

10. Chou TC, Scammell TE, Gooley JJ, Gaus SE, Saper CB, Lu J. Critical role of

dorsomedial hypothalamic nucleus in a wide range of behavioral circadian rhythms. J

Neurosci 2003;23:1069110702

11. Yoshida K, McCormack S, España RA, Crocker A, Scammell TE. Afferents to the orexin

neurons of the rat brain. J Comp Neurol 2006;494:845861

20 Page 21 of 42 Diabetes

12. Zhang S, Zeitzer JM, Yoshida Y, et al. Lesions of the suprachiasmatic nucleus eliminate

the daily rhythm of hypocretin1 release. Sleep 2004;27:619627

13. Zeitzer JM, Buckmaster CL, Parker KJ, Hauck CM, Lyons DM, Mignot E. Circadian and

homeostatic regulation of hypocretin in a primate model: implications for the

consolidation of wakefulness. J Neurosci 2003;23:35553560

14. Desarnaud F, MurilloRodriguez E, Lin L, et al. The diurnal rhythm of hypocretin in

young and old F344 rats. Sleep 2004;27:851856

15. Yamamoto Y, Ueta Y, Date Y, et al. Down regulation of the preproorexin gene

expression in genetically obese mice. Mol Brain Res 1999;65:1422

16. Sakurai T. The neural circuit of orexin (hypocretin): maintaining sleep and wakefulness.

Nat Rev Neurosci 2007;8:171181

17. Tsuneki H, Murata S, Anzawa Y, et al. Agerelated insulin resistance in hypothalamus and

peripheral tissues of orexin knockout mice. Diabetologia 2008;51:657667

18. Hara J, Yanagisawa M, Sakurai T. Difference in obesity phenotype between orexin

knockout mice and orexin neurondeficient mice with same genetic background and

environmental conditions. Neurosci Lett 2005;380:239242

19. Poli F, Plazzi G, Di Dalmazi G, et al. Body mass indexindependent metabolic alterations

in narcolepsy with cataplexy. Sleep 2009;32:14911497

20. Yi CX, Serlie MJ, Ackermans MT, et al. A major role for perifornical orexin neurons in

the control of glucose metabolism in rats. Diabetes 2009;58:19982005

21. Shiuchi T, Haque MS, Okamoto S, et al. Hypothalamic orexin stimulates feeding

associated glucose utilization in skeletal muscle via sympathetic nervous system. Cell

Metab 2009;10:466480

22. Tsuneki H, Wada T, Sasaoka T. Role of orexin in the central regulation of glucose and

energy homeostasis. Endocr J 2012;59:365374

21 Diabetes Page 22 of 42

23. Mieda M, Hasegawa E, Kisanuki YY, Sinton CM, Yanagisawa M, Sakurai T. Differential

roles of orexin receptor1 and 2 in the regulation of nonREM and REM sleep. J

Neurosci 2011;31:65186526

24. Hiramoto T1, Chida Y, Sonoda J, Yoshihara K, Sudo N, Kubo C. The hepatic vagus nerve

attenuates Fasinduced apoptosis in the mouse liver via α7 nicotinic acetylcholine

receptor. Gastroenterology 2008;134,21222131

25. Yonezawa R, Wada T, Matsumoto N, et al. Central versus peripheral impact of estradiol

on the impaired glucose metabolism in ovariectomized mice on a highfat diet. Am J

Physiol Endocrinol Metab 2012;303:E445E456

26. Kimura K, Yamada T, Matsumoto M, et al. Endoplasmic reticulum stress inhibits STAT3

dependent suppression of hepatic gluconeogenesis via dephosphorylation and

deacetylation. Diabetes 2012;61:6173

27. Takasaki I, Taniguchi K, Komatsu F, et al. Contribution of spinal galectin3 to acute

herpetic allodynia in mice. Pain 2012;153:585592

28. Ikeda O, Ozaki M, Murata S, et al. Autonomic regulation of liver regeneration after partial

hepatectomy in mice. J Surg Res 2009;152: 218223

29. Cretenet G, Le Clech M, Gachon F. Circadian clockcoordinated 12 period rhythmic

activation of the IRE1α pathway controls lipid metabolism in mouse liver. Cell Metab

2010;11:4757

30. Lee J, Sun C, Zhou Y, et al. p38 MAPKmediated regulation of Xbp1s is crucial for

glucose homeostasis. Nat Med 2011;17:12511260

31. Boden G, Chen X, Urbain JL. Evidence for a circadian rhythm of insulin sensitivity in

patients with NIDDM caused by cyclic changes in hepatic glucose production. Diabetes

1996;45,10441050

22 Page 23 of 42 Diabetes

32. Cailotto C1, van Heijningen C, van der Vliet J, et al. Daily rhythms in metabolic liver

and plasma glucose require a balance in the autonomic output to the liver.

Endocrinology 2008;149:19141925

33. Date Y, Ueta Y, Yamashita H, et al. Orexins, orexigenic hypothalamic peptides, interact

with autonomic, neuroendocrine and neuroregulatory systems. Proc Natl Acad Sci USA

1999;96:748753

34. Date Y, Mondal MS, Matsukura S, Nakazato M. Distribution of orexinA and orexinB

(hypocretins) in the rat spinal cord. Neurosci Lett 2000;288:8790

35. Hwang LL, Chen CT, Dun NJ. Mechanisms of orexininduced depolarizations in rat

dorsal motor nucleus of vagus neurones in vitro. J Physiol 2001;537:511520

36. van den Top M, Nolan MF, Lee K, et al. Orexins induce increased excitability and

synchronisation of rat sympathetic preganglionic neurones. J Physiol 2003;549:809821

37. Tanida M, Niijima A, Shen J, et al. Dosedifferent effects of orexinA on the renal

sympathetic nerve and blood pressure in urethaneanesthetized rats. Exp Biol Med

(Maywood) 2006;231:16161625

38. Yamanaka A, Beuckmann CT, Willie JT, et al. Hypothalamic orexin neurons regulate

arousal according to energy balance in mice. Neuron 2003;38:701713.

39. Goncalves AC, Tank J, Diedrich A, et al. Diabetic hypertensive leptin receptordeficient

db/db mice develop cardioregulatory autonomic dysfunction. Hypertension 2009;53:387

392

40. Mizuno M, Kamiya A, Kawada T, et al. Accentuated antagonism in vagal heart rate

control mediated through muscarinic potassium channels. J Physiol Sci 2008;58:381388.

41. Marcus JN, Aschkenasi CJ, Lee CE, et al. Differential expression of orexin receptors 1

and 2 in the rat brain. J Comp Neurol 2001;435:625

23 Diabetes Page 24 of 42

42. DeFronzo RA. Banting Lecture. From the triumvirate to the ominous octet: a new

paradigm for the treatment of type 2 diabetes mellitus. Diabetes 2009;58:773795.

43. Deng Y, Wang ZV, Tao C, et al. The Xbp1s/GalE axis links ER stress to postprandial

hepatic metabolism. J Clin Invest 2013;123:455468

44. Hotamisligil GS. Endoplasmic reticulum stress and the inflammatory basis of metabolic

disease. Cell 2010;140:900917

45. Lee J, Ozcan U. Unfolded protein response signaling and metabolic diseases. J Biol Chem

2014;289:12031211

46. DeVries JH. Glucose variability: where it is important and how to measure it. Diabetes

2013;62:14051408

47. Hatori M, Vollmers C, Zarrinpar A, et al. Timerestricted feeding without reducing caloric

intake prevents metabolic diseases in mice fed a highfat diet. Cell Metab 2012;15:848

860

48. Tabas I, Ron D. Integrating the mechanisms of apoptosis induced by endoplasmic

reticulum stress. Nat Cell Biol 2011;13:184190

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Figure legends

Figure 1. Daily rhythms in blood glucose levels were disrupted in Orexin/ mice. Male

Orexin/ mice (closed symbols) and their WT mice (open symbols) at 3 months old (A, C, D,

G, H, K, L) and 6 months old (B, E, F, I, J, M, N) were fed ad libitum. A and B: Daily changes

in blood glucose levels. n=6. CF: Food intake (C and E) and locomotor activity (D and F).

n=69. GJ: Daily changes in serum levels of insulin (G and I) and glucagon (H and J). n=39

for each time point. KN: The mRNA levels of genes related to hepatic gluconeogenesis,

Pgc1α (K), G6Pase (L) at 3 months old and Pepck (M) and G6Pase (N) at 6 months old in the

liver under ad libitum fed conditions. The expression levels were calculated as a ratio to those

in WT mice at ZT 8. n=627 for each time point. Data are the means ± S.E.M. ZT, Zeitgeber

time. *P<0.05, **P<0.01, and †P<0.1 vs. WT mice. aP<0.05 vs. glucose levels in WT mice at

ZT5. bP<0.05 vs. glucose levels in Orexin/ mice at ZT5.

Figure 2. Orexin A promoted to generate a daily rhythm in blood glucose levels in mice.

Orexin A (OXA, closed symbols) or saline (open symbols) was intracerebroventricularly

administered in mice. Arrows indicate the administration time. A: Effects of orexin A (3

nmol) or saline on blood glucose levels in 8weekold C57BL/6J mice fed ad libitum until

administration. n=4. B: Inhibitory effect of phenoxybenzamine (POB, 10 mg/kg) and

propranolol (PRO, 10 mg/kg) on the ICVorexin A (3 nmol)induced glucose elevation in

C57BL/6J mice. POB and PRO were intraperitoneally injected 10 min before the orexin

administration. The mice were fed until drug treatment. n=4. C: Effects of ICVorexin A (3

nmol) or saline on HGP rates before and during steadystate hyperinsulinemiceuglycemic

clamp in C57BL/6J mice fasted overnight. n=4. D and E: Effects of nighttime ICV

administration of orexin A (D, 0.3 nmol; E, 3 nmol) or saline at ZT 14 on blood glucose

levels in db/db (circle) and m+/m+ mice (triangle) at 8 weeks old. The mice were deprived of

25 Diabetes Page 26 of 42

food 6 h before ICV administration. n=37. F: Effects of daily administration of orexin A (0.3 nmol, once a day for 3 days) or saline at ZT 14 on fed blood glucose levels in 8weekold db/db mice. n=57. GK: Effects of nighttime administration of orexin A (0.3 nmol, once a day for 3 days) or saline at ZT 14 on fed glucose levels (G) and body weight (H) measured immediately before each administration, or fasting glucose levels (I), serum insulin levels (J) and the mRNA levels of Pgc1α, Pepck, and G6Pase in the liver (K) at ZT 8 (under 6h fasting condition) after the last administration in 20weekold db/db mice. n=57. L: Effects of daytime administration of orexin A (0.3 nmol, once a day for 3 days) or saline at ZT 2 on blood glucose levels in 8weekold db/db mice. n=45. M and N: Effects of continuous ICV infusion of orexin A (0.3 nmol/day) on blood glucose levels (M) measured at ZT 14 under fed conditions, and the mRNA levels of Pepck and G6Pase in the liver (N) at ZT 8 (after 6h fasting at the end of the infusion) in 8weekold db/db mice. Horizontal bar indicates the period of orexin A infusion. n=45. Data are the means ± S.E.M. *P<0.05, **P<0.01, and

†P<0.1 vs. saline control. aP<0.05, difference between the responses to saline and orexin A in the presence of POB and PRO. bP<0.05 and bbP<0.01, significant inhibition of the orexin A response by POB and PRO.

Figure 3. Nighttime administration of orexin A lowered daytime blood glucose via the parasympathetic pathway in mice. A: Effects of nighttime intraperitoneal injection of adrenaline (closed circle, 0.05 or 0.1 mg/kg) or saline (open circle) at ZT 14 on fed blood glucose levels in 8weekold db/db mice. n=3. Arrows indicate the administration time. BE:

Influence of hepatic parasympathectomy (HPx) on the orexin Ainduced changes in blood glucose levels. C57BL6J mice were shamoperated (B and C) or parasympathectomized (D and E). Orexin A (OXA, closed circle) or saline (open circle) was intracerebroventricularly administered at ZT 14 (once a day for 3 days), and blood glucose levels were measured under

26 Page 27 of 42 Diabetes

ad lib fed condition (B and D). Pyruvate tolerance tests were performed at ZT 8 (under 6h

fasting condition) after the last administration (C and E). n=37. Data are the means ± S.E.M.

*P<0.05, **P<0.01, and †P<0.1 vs. saline control in each genotype.

Figure 4. Bidirectional effects of orexin A on the regulation of hepatic gluconeogenesis in

mice. Orexin A (OXA) was intracerebroventricularly administered in mice under overnight

fasting state. In experiments to analyze hepatic gene expression profiles, the liver was isolated

at 120 min. A and B: The levels of blood glucose (A) and serum insulin (B) in 8weekold

C57BL/6J mice treated with ICVorexin A (closed triangle, 3 fmol; closed square, 3 nmol) or

ICVsaline (open circle). n=46 per group. C and D: Effects of ICVorexin A (3 fmol, 3 pmol

and 3 nmol) on the mRNA levels of Pepck (C) and G6Pase (D) in the liver of C57BL/6J mice.

n=1316. E and F: Effects of ICVorexin A (3 fmol and 3 nmol) on hepatic Pepck mRNA

levels in Ox1r/ (E) and Ox2r/ mice (F). n=912. G: Changes in the hepatic Pepck levels by

ICVorexin A (3 nmol) or ICVsaline administered 30 min after intraperitoneal injection of

phenoxybenzamine (POB, 5 mg/kg) or saline into C57BL/6J mice. n=1425. H: Changes in

the Pepck levels by ICVorexin A (3 fmol) or ICVsaline administered 30 min after

intraperitoneal injection of atropine (ATR, 2 mg/kg) or saline into C57BL/6J mice. n=1630.

Data are expressed as % (CF) or delta% (G and H) of saline control. *P<0.05 and **P<0.01.

Figure 5. Agerelated development of hepatic insulin resistance in Orexin/ mice. A and B:

Pyruvate tolerance test performed in male (A) or female (B) WT mice (open circle) and

Orexin/ mice (closed circle) at 6 months old under overnight fasting condition. n=67. C:

Insulininduced phosphorylation of Akt at Ser473 in the liver and skeletal muscle of male WT

and Orexin/ mice at 6 months old. The tissues were isolated 15 min after intravenous

injection of insulin (1 Unit/kg) under overnight fasting conditions. n=45. D and E: The

27 Diabetes Page 28 of 42

mRNA (upper, n=1415) and protein (lower, n=610) levels of IRS1 (D) and IRS2 (E) in the liver of WT and Orexin/ mice at 6 months old under fed or overnight fasting conditions. Data are the means ± S.E.M. *P<0.05, **P<0.01, and †P<0.1. A.U., arbitrary unit.

Figure 6. Enhancement of ER stress in the liver of Orexin/ mice. A: The mRNA levels of

Chop in the liver of WT and Orexin/ mice at 6 months old under 5h fasting conditions. B and C: Influence of 1h refeeding after 24h fasting period on the phosphorylation levels of

IRE1α at Ser724 (B) and JNK at Thr183/Tyr185 (C) in the liver of 6monthold WT and Orexin/ mice. D: The phosphorylation levels of JNK at Thr183/Tyr185 in the liver of 4monthold WT,

Ox1r/, and Ox2r/ mice that were refed for 1 h after 24h fasting period. Data are the means ±

S.E.M. of 4 to 7 mice. *P<0.05 and **P<0.01. A.U., arbitrary unit. E: Electron microscopy of liver sections from WT (a, b) and Orexin/ mice (c, d) at 6 months old. The liver tissues were dissected at ZT 8 under fed condition. Images were obtained at 3,000x (a, c) or 8,000x magnification (b, d). Scale bars: 2 m (a, c) and 0.5 m (b, d). F: Putative mechanism by which orexin regulates daily rhythm in HGP. Under SCN control, central orexin levels increase during wakefulness. Orexin A enhances HGP via OX2R and sympathetic (SNS) pathway, and then suppressed it via OX1R and parasympathetic (PNS) pathway. These cause a daily blood glucose oscillation that appears to be required for maintenance of energy homeostasis and prevention of hepatic insulin resistance.

28 Page 29 of 42 Diabetes

3 months old 6 months old AB 130 a 120 * * ** a 120 * 110 * 110 100 a

100 90 WT b 90 Orexin-/- 80 Blood glucose (mg/dL) Blood glucose (mg/dL) 0

5 8 11 14 17 20 23 2 5 8 11 14 17 20 23 2 ZT ZT

CD40 E 40 F 30 2 30 2 20 20 1 1 counts/2h) * counts/2h) 5

* 5 10 * * * * 10 * * *** * * * * * † * **† † Food intake Locomotor

Food intake * (mg/g mouse) Locomotor * * (mg/g mouse) * 0 0 * 0 0 * (x 10 (x

* 10 (x 0112 24 2240112 24 224 0112 24 2240112 24 224 ZT ZT ZT ZT G HIJ * 8 0.3 8 0.3 6 6 0.2 0.2 4 ** 4 2 0.1 2 0.1

Insulin (ng/mL) 0 0 Insulin (ng/mL) 0 0 Glucagon (ng/mL) 2 8 14 20 2 8 14 20 2 8 14 20 Glucagon (ng/mL) 2 8 14 20 ZT ZT ZT ZT K L M N 6 2 ** 6 † 1 4 4 1 mRNA (fold) mRNA (fold) mRNA (fold) 2 * mRNA (fold) 2

α * 0 0 * 0 0 Pepck Pgc1 2 8 14 20 G6Pase 2 8 14 20 2 8 14 20 G6Pase 2 8 14 20 ZT ZT ZT ZT

Fig. 1 Diabetes Page 30 of 42

A C57BL/6J B C57BL/6J C C57BL/6J saline 250 POB+PRO OXA OXA ** ** ** 300 200 30 ** bb b * 150 a 20 200 * 100 saline/ saline 10 100 saline/ OXA HGP (mg/kg·min)

Glucose (mg/dL) 50 POB+PRO/ saline

Glucose (mg/dL) POB+PRO/ OXA 0 0 0 OXA - + - + 0 60 120 180 240 -10 0 30 60 120 Time (min) Time (min) basal clamp

DEdb/db db/db F db/db saline saline OXA OXA 400 400 (0.3 nmol) 400 ** * (3 nmol) m+/m+ m+/m+ 300 300 saline 300 saline OXA OXA 200 200 (0.3 nmol) 200 (3 nmol) ** * 100 saline 100 * * * * 100 † Glucose (mg/dL) OXA (0.3 nmol) Glucose (mg/dL) Glucose (mg/dL) 0 0 0 012 6 12 012 6 12 14 20 28 14 20 28 14 220 Time (h) Time (h) ZT

G db/db H db/db I db/db J db/db K db/db Pgc1α Pepck G6Pase * 10 150 80 400 400 8 * ** ** 60 300 * 300 6 100 200 * 40 200 4 50 saline 20 saline

100 100 mRNA (%) OXA OXA 2 Insulin (ng/mL) Body weight (g) weight Body Glucose (mg/dL) 0 0 Glucose (mg/dL) 0 0 0 012 012 OXA + OXA + OXA + + + Time (day) Time (day) - - - - -

L db/db MNdb/db db/db Pepck G6Pase 500 * 400 400 150 300 * 300 100 200 200 saline saline 100 100 50

OXA OXA mRNA (%) Glucose (mg/dL) * Glucose (mg/dL) 0 0 0 2142142142 -2 -1 0 1 2 3 4 5 6 7 OXA - + - + ZT Time (day)

Fig. 2 Page 31 of 42 Diabetes

A adrenaline (0.05 mg/kg) adrenaline (0.1 mg/kg)

600 500 * * *** 400 * * 300 200 saline Glucose (mg/dL) 100 db/db adrenaline 0 14 2 14 2 14 2 14 2 14 2142 14 ZT

B 160 C 140 * * 160 120 * * ** 140 100 120 100 † 80 80 60 * 60 * 40 saline ** 40 Glucose (mg/dL) 20 Sham-operated OXA Glucose (mg/dL) 20 Sham-operated 0 0 14 20 28 14 20 28 14 220 0 15 30 60 120 Time (min) Time (min) DE 160 140 ** * * 160 120 ** 140 ** 100 120 100 80 80 60 60 40 40 Glucose (mg/dL) 20 HPx Glucose (mg/dL) 20 HPx 0 0 14 20 28 14 20 28 14 220 0 15 30 60 120 Time (min) Time (min)

Fig. 3 Diabetes Page 32 of 42

ABsaline OXA (3 fmol) 600 200 OXA (3 nmol) * 400

100 200

* Insulin (pg/mL) Glucose (mg/dL) 0 0 0 60 120 240 0 60 120 240 Time (min) Time (min) CD 200 ** ** * 200 * mRNA (%) mRNA (%) 100 100 Pepck

0 G6Pase 0 saline saline 3 fmol 3 fmol 3 nmol 3 pmol 3 nmol 3 pmol OXA OXA E Ox1r-/- F Ox2r-/- 140 * * 120 120 * 100 100 80 80 60 mRNA (%) 60 mRNA (%) 40 40 20 20 Pepck 0 Pepck 0 saline saline 3 fmol 3 fmol 3 nmol 3 nmol OXA OXA

GH30 10 %)

* %) ∆ 20 ∆ 0

10 -10 mRNA ( mRNA ( 0 -20 Pepck -10 Pepck * OXA - + - + OXA - + - + POB - - + + ATR - - + +

Fig. 4 Page 33 of 42 Diabetes

A B 400 400 * 300 * 300 † ** * 200 ** 200

100 100 male female Glucose (mg/dL) 0 0 0 30 60 90 120 0306090120 Time (min) Time (min)

C Liver Skeletal muscle p-Akt Akt 4 ** 4 ** 3 * 3 2 2 1 1

p-Akt/Akt (A.U.) 0 0 Insulin - + - + - + - + WT Orexin-/- WT Orexin-/- D E 2 2 * 1 1 mRNA (A.U.) mRNA (A.U.) 0 0 -/- -/- -/- -/- Irs2 Irs1 WT Orexin WT Orexin WT Orexin WT Orexin Fed Fasting Fed Fasting

WT Orexin-/- WT Orexin-/- IRS1 IRS2 -actin -actin

1 * 1 * (A.U.) (A.U.) 0 0 IRS1 protein WT Orexin-/- IRS2 protein WT Orexin-/- Fed Fed

Fig. 5 Diabetes Page 34 of 42

A B WT Orexin-/-

* p-IRE1 2 IRE1 *

 2 1 /IRE1 mRNA (A.U.)

 1 (A.U.) Chop

0 p-IRE1 0 WT Orexin-/- WT Orexin-/-

C D C57BL/6J Ox1r -/- Ox2r -/- WT Orexin-/- refed (h) 0 10101

p-JNK p-JNK

JNK JNK 2 * ** ** 2 * *

1 1

0 (A.U.) p-JNK/JNK p-JNK/JNK (A.U.) p-JNK/JNK -/- 0 WT Orexin refed (h) 010101 C57BL/6J Ox1r -/- Ox2r -/-

E WT Orexin-/- F SCN ac Orexin A

OX1R OX2R

SNS PNS

HGP rhythm bd

Glucose supply ER maintenance

Energy Insulin homeostasis resistance

Fig. 6 Page 35 of 42 Diabetes

Supplementary Table 1. Primers for real-time PCR.

Genes Forward Primer Reverse Primer Pgc1a GCCCGGTACAGTGAGTGTTC CTGGGCCGTTTAGTCTTCCT Pepck CAGGATCGAAAGCAAGACAGT AAGTCCTCTTCCGACATCCAG G6Pase GAAAAAGCCAACGTATGGATTCC CAGCAAGGTAGATCCGGGA Insulin receptor AATGGCAACATCACACACTACC CAGCCCTTTGAGACAATAATCC Irs1 CTCTACACCCGAGACGAACAC TGGGCCTTTGCCCGATTATG Irs2 GGAGAACCCAGACCCTAAGCT GATGCCTTTGAGGCCTTCAC Chop CCACCACACCTGAAAGCAGAA AGGTGAAAGGCAGGGACTCA Tnfa AAGCCTGTAGCCCACGTCGTA GGCACCACTAGTTGGTTGTCTTTG IL1b CAACCAACAAGTGATATTCTCCATG GATCCACACTCTCCAGCTGCA Preproorexin AGACGACGGCCTCAGAC GGCCCAGGGAACCTTTG Per1 GCTTCGTGGACTTGACACCT TGCTTTAGATCGGCAGTGGT Per2 GTTCCAGGCTGTGGATGAA GGCGTCTCGATCAGATCCT S18 ribosomal protein AGTTCCAGCACATTTTGCGAG TCATCCTCCGTGAGTTCTCCA

Diabetes Page 36 of 42

Supplementary Table 2

List of top biofunctions altered in the liver of male orexin knockout mice at 6 months old, based on the GeneChip and IPA analyses.

Associated Category Function Annotation p-value Gene Symbol genes

1. Diseases and Disorders

Cancer 2.44E-10 to 7.24E-03 536 Endocrine System Disorders 9.69E-06 to 2.37E-03 161 Gastrointestinal Disease 9.69E-06 to 5.80E-03 264 Metabolic Disease 9.69E-06 to 5.46E-03 136

ADRA1B, ADRA2A, ADRA2C, ANKRD44, AR, ATF6, ATP10A, ATP4A, ATXN2, BCL2L11, BTC, CA14, CACNA1S, CCDC68, CCND2, CCR2, CD274, CD28, CD40LG, CD80 (includes EG:12519), COL27A1, COL4A1, DAB1, DCAKD, DDAH1, DHX16, DPP4, DUSP1, EDNRA, ENPP1, ERBB3 (includes EG:13867), FKBP15, FRMD4B, GABRA3, GFAP, GRIA4, GSK3B, HGF, HIST1H3A (includes others), HLA-C, HLA-DOB, HLA-DRA, HNF1A, HOXA3, HSPA1A/HSPA1B, ICA1, ICOS, ID1, IGF1R, IGFBP7, IGHM, IL1A, IL1RN, diabetes mellitus 9.69E-06 110 JUN, KCNJ6, KIF11, Klrb1c (includes others), LCN2, LEPR, MPG, MSH5 (includes EG:17687), MT1E, NAGLU, NOTCH4, NR3C2 (includes EG:110784), NR4A2, NSF, PDCD4, PDE4B, PDE7A, PDE8A, PDGFRL, PGM1, PON2, PPP1R11, PPP3CA, PPP3CB, PRDM10, PRF1, PRKCE, PRKDC, PSMB8, PSMC2, PTPN2, PTTG1, RNF220, RSAD2, RSBN1, Scd2, SCN7A, SELL, SIAE, SLC6A2, SLC9A9, SPRED1, STAT3, TAP1, THRB, TIMP1, TLR4, TMEFF2, TRIM10, TRIM40, USP7, VARS2, VDR, WAC, ZBTB16, ZEB2, ZKSCAN3

ADRA1B, ADRA2A, ADRA2C, AKT3, ANKRD44, AR, ARX, ATF6, ATP10A, ATP4A, ATXN2, BCL2L11, BTC, CA14, CACNA1S, CCDC68, CCND2, CCR2, CD274, CD28, CD36, CD40LG, CD80 (includes EG:12519), CDKN1A, COL27A1, COL4A1, CPE (includes EG:12876), DAB1, DCAKD, DDAH1, DHX16, DPP4, DUSP1, EDNRA, ENPP1, ERBB3 (includes EG:13867), FGF23, FGFR2, FKBP15, FRMD4B, GABRA3, GFAP, GRIA4, GSK3B, HELLS, HGF, HIST1H3A (includes others), HLA-C, HLA-DOB, HLA-DRA, HNF1A, HOXA3, HSPA1A/HSPA1B, HTR1A, ICA1, ICOS, ID1, IGF1R, IGFBP1 (includes EG:16006), IGFBP7, IGHM, IL1A, IL1RN, JUN, KCNJ6, KIF11, Klrb1c (includes others), LCN2, glucose metabolism disorder 1.15E-05 134 LEPR, LIPA, LMNA, MARK2, MPG, MSH5 (includes EG:17687), MT1E, NAGLU, NOTCH4, NPY1R, NR3C2 (includes EG:110784), NR4A2, NSF, PDCD4, PDE4B, PDE7A, PDE8A, PDGFRL, PGM1, PON2, PPARGC1A, PPP1R11, PPP3CA, PPP3CB, PRDM10, PRF1, PRKAB2, PRKCE, PRKDC, PRLR, PSMB8, PSMC2, PTPN2, PTTG1, PYCARD, RNF220, RSAD2, RSBN1, Scd2, SCN7A, SELL, SIAE, SLC6A2, SLC6A6, SLC9A9, SPRED1, STAT3, STEAP2, TAP1, THRB, TIMP1, TLR3, TLR4, TMEFF2, TRAF3, TRIB3, TRIM10, TRIM40, USP7, VARS2, VDR, WAC, WRN, ZBTB16, ZEB2, ZKSCAN3

ATXN2, CCR2, CD274, CD28, CD40LG, CD80 (includes EG:12519), COL27A1, DHX16, ERBB3 (includes EG:13867), HIST1H3A (includes others), HLA-C, HLA-DOB, HLA-DRA, insulin-dependent diabetes mellitus 9.28E-04 43 HNF1A, HSPA1A/HSPA1B, ICA1, ICOS, IGFBP7, IGHM, IL1RN, MPG, MSH5 (includes EG:17687), NOTCH4, PDCD4, PGM1, PPP1R11, PPP3CA, PPP3CB, PRF1, PRKDC, PSMB8, PTPN2, RSBN1, SELL, SIAE, SPRED1, TAP1, TRIM10, TRIM40, USP7, VARS2, VDR, ZKSCAN3

experimentally-induced diabetes 2.37E-03 17 CCND2, COL4A1, DDAH1, DUSP1, GFAP, HLA-C, ID1, IL1RN, JUN, LCN2, MT1E, NSF, PRKCE, PSMC2, RSAD2, SCN7A, TIMP1 Hematological Disease 1.64E-05 to 6.43E-03 123

2. Molecular and Cellular Functions

Gene 1.13E-11 to 1.99E-03 346 Expression

ABLIM1, ACTR3, ACVR1C, AEBP2, AFAP1L2, AFF1, AKAP13, ANP32A, APBB2, AR, ARHGEF11, ARID4B, ARNT, ARNTL, ASPM, ATF6, ATF7IP, ATP2C1, ATRX, ATXN3, BACH2, BAZ2A, BCLAF1, BEX1, BRD8, BRWD1, BTRC, C1D, CAMK2D, CAMK4, CAND1, CAPRIN2, CASK, CBFA2T2, CBX1, CBX2, CCNE1, CCNT2, CD28, CD40LG, CDC73 (includes EG:214498), CDK5RAP2, CDKN1A, CHD1, CHD2, CHD8, CHEK2, CHST1, CISH, CITED2, COPS2, CRTC2, CUX1, DDX58, DUSP1, DYRK1A, EAF2, EBF1, EGF (includes EG:13645), EGR1, ENO1, EPC1, ETS2, F2RL1, FGF23, FGFR2, FOXK1, FOXK2, FOXP2, FOXP4, FUBP1, FZD1, FZD2, FZD8, GAB2, GABPB1, GADD45G, GCM1, Gm4924, GPR39, GSK3B, GTF2A1, GTF3C1, GTF3C6, HDAC6, HDAC9, HELLS, HGF, HIP1 (includes EG:176224), HIPK2, HIVEP1, HIVEP2, HIVEP3, HMGN3, HNF1A, HOXA2, HOXD13, HSF2, ID1, ID3 (includes EG:15903), Ifi204 (includes others), IL1A, ILF2, ILF3, ING5, INHBB, JARID2, JUN, JUNB, KAT2A, KCTD1, KCTD13, KDM1A, KDM5B, KDM5C, KHDRBS1, KLF12, KLF6, KLF7, KLF9, LBH, LCORL, LCP2, LEPR, LIME1, LMNA, LPAR1, LPIN2, LRP6, LYL1, LZTR1, MAF, MAGI2, MAML1, transcription 1.13E-11 277 MAP2K7, MED12, MED13, MED14, MED15, MET, MLF1, MPG, MSX1, MTERFD3, MTPN, MX1, MYCBP, MYNN, MYPOP, NCAM1, NCOA7, NCOR1, NDN, NEK6, NFE2L2, NFIA, NFIB, NFIC, NKAP, NOTCH4, NR2C1, NR2C2, NR2F2, NR3C2 (includes EG:110784), NR4A2, NRF1, NRIP1, PAK6, PARP1, PAX9, PDCD4, PER3, PHF21A, PHGDH, PHIP, PKNOX2, PLEKHG2, PLK3, PMF1, POU2AF1, POU2F1, POU6F1, PPARGC1A, PPM1D, PPP2R5D, PPP3CA, PRKCE, PRKCZ, PRKDC, PRLR, PROP1, PSIP1, PTMS, PTPRK, PTTG1, PVT1, RAD21, RAF1, RARB, RBBP8, RBM14, RBM39, RBX1, RCAN1, RCOR1, RECQL5, RFX4, RFX5, RNF20, RNPS1, RORA, RXRG, SALL1, SCAF8, Scd2, SIN3A, SIRT4, SMARCAL1, SMYD1, SNAI2, SNX6, SOX11, SOX12, SOX17, SOX21, SOX5, SP2, SP7, SPEN, SS18, STAT1, STAT2, STAT3, STAT5A, STRAP, SYK, TAF3 (includes EG:209361), TAF5, TAF6L, TARDBP, TBL1XR1, TBX2, TBX4, TCEB3, TCF7, THRAP3, THRB, TIMP1, TLE4, TLR3, TLR4, TOP2A, TRAF3, TRIB3, TRIP11, TRRAP, TSHZ3, UBE3A, USP47, VAV1, VAV2, VAV3, VDR, WNT5A, WRN, XAB2, YEATS2, ZBTB16, Zfp53, Zfp68, ZHX3, ZKSCAN3, ZNF143, ZNF292, ZNF295, ZNF440/ZNF808, ZNF593

ABLIM1, ACTR3, ACVR1C, AEBP2, AFAP1L2, AFF1, ANP32A, APBB2, AR, ARHGEF11, ARID4B, ARNT, ARNTL, ASPM, ATF6, ATF7IP, ATP2C1, ATRX, ATXN3, BACH2, BAZ2A, BCLAF1, BEX1, BRD8, BRWD1, BTRC, C1D, CAMK2D, CAMK4, CAND1, CAPRIN2, CASK, CBFA2T2, CBX1, CBX2, CCNE1, CCNT2, CD28, CD40LG, CDC73 (includes EG:214498), CDK5RAP2, CDKN1A, CHD1, CHD2, CHD8, CHEK2, CISH, CITED2, COPS2, CRTC2, CUX1, DDX58, DUSP1, DYRK1A, EAF2, EBF1, EGF (includes EG:13645), EGR1, ENO1, EPC1, ETS2, F2RL1, FGF23, FGFR2, FOXK1, FOXK2, FOXP2, FOXP4, FUBP1, FZD1, FZD2, FZD8, GAB2, GABPB1, GADD45G, GCM1, Gm4924, GPR39, GSK3B, GTF2A1, GTF3C1, GTF3C6, HDAC6, HDAC9, HGF, HIP1 (includes EG:176224), HIPK2, HIVEP1, HIVEP2, HIVEP3, HMGN3, HNF1A, HOXA2, HOXD13, HSF2, ID1, ID3 (includes EG:15903), Ifi204 (includes others), IL1A, ILF2, ILF3, ING5, INHBB, JARID2, JUN, JUNB, KAT2A, KCTD1, KCTD13, KDM1A, KDM5B, KDM5C, KHDRBS1, KLF12, KLF6, KLF7, KLF9, LBH, LCORL, LCP2, LEPR, LIME1, LMNA, LPAR1, LPIN2, LRP6, LYL1, LZTR1, MAF, MAGI2, MAML1, MAP2K7, MED12, transcription of RNA 2.68E-11 271 MED13, MED14, MED15, MET, MLF1, MPG, MSX1, MTERFD3, MTPN, MYCBP, MYNN, MYPOP, NCAM1, NCOA7, NCOR1, NDN, NEK6, NFE2L2, NFIA, NFIB, NFIC, NKAP, NOTCH4, NR2C1, NR2C2, NR2F2, NR3C2 (includes EG:110784), NR4A2, NRF1, NRIP1, PAK6, PARP1, PAX9, PDCD4, PER3, PHF21A, PHGDH, PHIP, PKNOX2, PLEKHG2, PLK3, PMF1, POU2AF1, POU2F1, POU6F1, PPARGC1A, PPM1D, PPP2R5D, PPP3CA, PRKCE, PRKCZ, PRKDC, PRLR, PROP1, PSIP1, PTMS, PTPRK, PTTG1, RAD21, RAF1, RARB, RBBP8, RBM14, RBM39, RBX1, RCAN1, RCOR1, RECQL5, RFX4, RFX5, RNF20, RNPS1, RORA, RXRG, SALL1, SCAF8, Scd2, SIN3A, SMARCAL1, SMYD1, SNAI2, SNX6, SOX11, SOX12, SOX17, SOX21, SOX5, SP2, SP7, SPEN, SS18, STAT1, STAT2, STAT3, STAT5A, STRAP, SYK, TAF3 (includes EG:209361), TAF5, TAF6L, TARDBP, TBL1XR1, TBX2, TBX4, TCEB3, TCF7, THRAP3, THRB, TIMP1, TLE4, TLR3, TLR4, TOP2A, TRAF3, TRIB3, TRIP11, TRRAP, TSHZ3, UBE3A, USP47, VAV1, VAV2, VAV3, VDR, WNT5A, WRN, XAB2, YEATS2, ZBTB16, Zfp53, Zfp68, ZHX3, ZKSCAN3, ZNF143, ZNF292, ZNF295, ZNF440/ZNF808, ZNF593

ABLIM1, AEBP2, AFAP1L2, AFF1, APBB2, AR, ARHGEF11, ARNT, ARNTL, ATF6, ATF7IP, ATRX, BACH2, BCLAF1, BEX1, BRD8, BRWD1, BTRC, C1D, CAMK4, CAND1, CAPRIN2, CASK, CBFA2T2, CBX1, CBX2, CD28, CDC73 (includes EG:214498), CDK5RAP2, CHD1, CHD2, CHD8, CHEK2, CITED2, COPS2, CUX1, DDX58, EAF2, EBF1, EGF (includes EG:13645), EGR1, ENO1, EPC1, ETS2, F2RL1, FGF23, FGFR2, FOXK1, FOXK2, FOXP2, FOXP4, FUBP1, FZD1, FZD2, FZD8, GABPB1, GCM1, Gm4924, GSK3B, GTF2A1, GTF3C1, GTF3C6, HDAC6, HDAC9, HIPK2, HIVEP1, HIVEP2, HIVEP3, HMGN3, HNF1A, HOXA2, HOXD13, HSF2, ID1, ID3 (includes EG:15903), Ifi204 (includes others), IL1A, ILF2, ILF3, ING5, JARID2, JUN, JUNB, KAT2A, KCTD1, KCTD13, KDM1A, KDM5B, KHDRBS1, KLF12, KLF6, KLF7, KLF9, LBH, LCORL, LIME1, transcription of DNA 4.21E-09 214 LPIN2, LRP6, LYL1, LZTR1, MAF, MAML1, MED12, MED13, MED14, MET, MLF1, MSX1, MTERFD3, MYCBP, MYNN, MYPOP, NCOA7, NCOR1, NDN, NFE2L2, NFIA, NFIB, NFIC, NKAP, NOTCH4, NR2C1, NR2C2, NR2F2, NR3C2 (includes EG:110784), NR4A2, NRF1, NRIP1, PARP1, PAX9, PDCD4, PER3, PHF21A, PHIP, PKNOX2, PLK3, PMF1, POU2AF1, POU2F1, POU6F1, PPARGC1A, PPP2R5D, PPP3CA, PRKDC, PROP1, PSIP1, PTPRK, PTTG1, RAD21, RARB, RBBP8, RBM14, RBM39, RCAN1, RCOR1, RFX4, RFX5, RNF20, RNPS1, RORA, RXRG, SALL1, SCAF8, SIN3A, SMARCAL1, SMYD1, SNAI2, SNX6, SOX11, SOX12, SOX17, SOX21, SOX5, SP2, SP7, SPEN, SS18, STAT1, STAT2, STAT3, STAT5A, STRAP, TAF3 (includes EG:209361), TAF5, TAF6L, TARDBP, TBL1XR1, TBX2, TBX4, TCEB3, TCF7, THRAP3, THRB, TLE4, TLR3, TLR4, TOP2A, TRIP11, TRRAP, TSHZ3, UBE3A, USP47, VAV1, VDR, WNT5A, XAB2, YEATS2, ZBTB16, ZHX3, ZKSCAN3, ZNF143, ZNF292, ZNF295, ZNF593

ABLIM1, ACO1, ACTR3, ACVR1C, AEBP2, AFAP1L2, AFF1, ANP32A, APBB2, AR, ARHGEF11, ARID4B, ARNT, ARNTL, ASPM, ATF6, ATF7IP, ATP2C1, ATRX, ATXN3, BACH2, BAZ2A, BCLAF1, BEX1, BRD8, BRWD1, BTRC, C1D, CAMK2D, CAMK4, CAND1, CAPRIN2, CASK, CBFA2T2, CBX1, CBX2, CCNE1, CCNT2, CD28, CD40LG, CD47, CDC73 (includes EG:214498), CDK5RAP2, CDKN1A, CHD1, CHD2, CHD8, CHEK2, CISH, CITED2, COPS2, CRTC2, CUX1, DDX58, DHX58, DNAJC1, DUSP1, DYRK1A, EAF2, EBF1, EGF (includes EG:13645), EGR1, EIF2A, EIF2S2, EIF5B, ENO1, EPC1, ETF1, ETS2, F2RL1, FGF23, FGFR2, FOXK1, FOXK2, FOXP2, FOXP4, FUBP1, FZD1, FZD2, FZD8, GAB2, GABPB1, GADD45G, GCM1, Gm4924, GPR39, GSK3B, GTF2A1, GTF3C1, GTF3C6, HBP1, HDAC6, HDAC9, HGF, HIP1 (includes EG:176224), HIPK2, HIVEP1, HIVEP2, HIVEP3, HMGN3, HNF1A, HOXA10, HOXA2, HOXD13, HSF2, ID1, ID3 (includes EG:15903), Ifi204 (includes others), IL1A, ILF2, ILF3, ING5, INHBB, JARID2, JUN, JUNB, KAT2A, KCTD1, KCTD13, KDM1A, KDM5B, KDM5C, KHDRBS1, KLF12, KLF6, KLF7, KLF9, LBH, LCORL, LCP2, LEPR, LIME1, LMNA, LPAR1, LPIN2, LRP6, LYL1, LZTR1, MAF, MAGI2, MAML1, MAP2K4, MAP2K7, MAP4K4, MAP4K5, MBP, MED12, MED13, MED14, MED15, MET, MLF1, MPG, MRPL15 (includes EG:27395), expression of RNA 5.40E-11 291 MSX1, MTERFD3, MTPN, MYCBP, MYNN, MYPOP, NCAM1, NCOA7, NCOR1, NDN, NEK6, NFE2L2, NFIA, NFIB, NFIC, NKAP, NOTCH4, NR2C1, NR2C2, NR2F2, NR3C2 (includes EG:110784), NR4A2, NRF1, NRIP1, PAIP1, PAK1, PAK6, PARP1, PAX9, PDCD4, PER3, PHF21A, PHGDH, PHIP, PKNOX2, PLEKHG2, PLK3, PMF1, POU2AF1, POU2F1, POU6F1, PPARGC1A, PPM1D, PPP2R5D, PPP3CA, PRKCE, PRKCZ, PRKDC, PRLR, PROP1, PSIP1, PTMS, PTPRK, PTTG1, RAD21, RAF1, RARB, RBBP8, RBM14, RBM39, RBX1, RCAN1, RCOR1, RECQL5, RFX4, RFX5, RGS2 (includes EG:19735), RNF20, RNPS1, RORA, Rps24, RXRG, SALL1, SCAF8, Scd2, SIN3A, SMARCAL1, SMYD1, SNAI2, SNX6, SOX11, SOX12, SOX17, SOX21, SOX5, SP2, SP7, SPEN, SS18, STAT1, STAT2, STAT3, STAT5A, STRAP, SYK, TAF3 (includes EG:209361), TAF5, TAF6L, TARDBP, TBL1XR1, TBX2, TBX4, TCEB3, TCF7, THRAP3, THRB, TIMP1, TLE4, TLR3, TLR4, TOP2A, TRAF3, TRIB3, TRIP11, TRRAP, TSHZ3, UBE3A, USP47, VAV1, VAV2, VAV3, VCAM1, VDR, WNT5A, WRN, XAB2, YEATS2, ZBTB16, Zfp53, Zfp68, ZHX3, ZKSCAN3, ZNF143, ZNF292, ZNF295, ZNF440/ZNF808, ZNF593

ABLIM1, AEBP2, AFAP1L2, AFF1, APBB2, AR, ARHGEF11, ARNT, ARNTL, ATF6, ATF7IP, ATRX, BACH2, BCLAF1, BEX1, BRD8, BRWD1, BTRC, C1D, CAMK4, CAND1, CAPRIN2, CASK, CBFA2T2, CBX1, CBX2, CD28, CDC73 (includes EG:214498), CDK5RAP2, CHD1, CHD2, CHD8, CHEK2, CITED2, COPS2, CUX1, DDX58, EAF2, EBF1, EGF (includes EG:13645), EGR1, ENO1, EPC1, ETS2, F2RL1, FGF23, FGFR2, FOXK1, FOXK2, FOXP2, FOXP4, FUBP1, FZD1, FZD2, FZD8, GABPB1, GCM1, Gm4924, GSK3B, GTF2A1, GTF3C1, GTF3C6, HDAC6, HDAC9, HIPK2, HIVEP1, HIVEP2, HIVEP3, HMGN3, HNF1A, HOXA2, HOXD13, HSF2, ID1, ID3 (includes EG:15903), Ifi204 (includes others), IL1A, IL1RN, ILF2, ILF3, ING5, JARID2, JUN, JUNB, KAT2A, KCTD1, KCTD13, KDM1A, KDM5B, KHDRBS1, KLF12, KLF6, KLF7, KLF9, LBH, LCORL, expression of DNA 5.69E-09 215 LIME1, LPIN2, LRP6, LYL1, LZTR1, MAF, MAML1, MED12, MED13, MED14, MET, MLF1, MSX1, MTERFD3, MYCBP, MYNN, MYPOP, NCOA7, NCOR1, NDN, NFE2L2, NFIA, NFIB, NFIC, NKAP, NOTCH4, NR2C1, NR2C2, NR2F2, NR3C2 (includes EG:110784), NR4A2, NRF1, NRIP1, PARP1, PAX9, PDCD4, PER3, PHF21A, PHIP, PKNOX2, PLK3, PMF1, POU2AF1, POU2F1, POU6F1, PPARGC1A, PPP2R5D, PPP3CA, PRKDC, PROP1, PSIP1, PTPRK, PTTG1, RAD21, RARB, RBBP8, RBM14, RBM39, RCAN1, RCOR1, RFX4, RFX5, RNF20, RNPS1, RORA, RXRG, SALL1, SCAF8, SIN3A, SMARCAL1, SMYD1, SNAI2, SNX6, SOX11, SOX12, SOX17, SOX21, SOX5, SP2, SP7, SPEN, SS18, STAT1, STAT2, STAT3, STAT5A, STRAP, TAF3 (includes EG:209361), TAF5, TAF6L, TARDBP, TBL1XR1, TBX2, TBX4, TCEB3, TCF7, THRAP3, THRB, TLE4, TLR3, TLR4, TOP2A, TRIP11, TRRAP, TSHZ3, UBE3A, USP47, VAV1, VDR, WNT5A, XAB2, YEATS2, ZBTB16, ZHX3, ZKSCAN3, ZNF143, ZNF292, ZNF295, ZNF593

ABLIM1, AR, ARNT, ARNTL, ATF6, ATF7IP, BEX1, BRD8, BRWD1, CAPRIN2, CASK, CBFA2T2, CBX2, CD28, CDC73 (includes EG:214498), CHD1, CHD2, CHD8, CITED2, COPS2, CUX1, DDX58, EGR1, ENO1, EPC1, ETS2, F2RL1, FGFR2, FOXK1, FOXK2, FOXP2, FOXP4, FUBP1, FZD8, GABPB1, Gm4924, GTF2A1, GTF3C6, HDAC9, HIPK2, HMGN3, HNF1A, HOXA2, HOXD13, HSF2, ID1, ID3 (includes EG:15903), Ifi204 (includes others), IL1A, JARID2, JUN, JUNB, KAT2A, KDM1A, KLF12, KLF6, KLF7, KLF9, LCORL, LIME1, LPIN2, LRP6, MAF, MAML1, MED12, MED13, MED14, MET, MSX1, MYPOP, NCOA7, NCOR1, NFE2L2, NFIA, NFIB, NFIC, NR2C1, NR2C2, NR2F2, NR4A2, activation of DNA endogenous 4.17E-07 148 NRF1, NRIP1, PARP1, PAX9, PER3, PHF21A, PHIP, PKNOX2, PLK3, PMF1, POU2AF1, POU2F1, PPARGC1A, PPP2R5D, PPP3CA, PRKDC, PROP1, PSIP1, PTTG1, RAD21, RARB, RBBP8, RBM14, RCOR1, RFX4, RFX5, RORA, RXRG, SALL1, SCAF8, SIN3A, SMARCAL1, SNAI2, SOX11, SOX12, SOX17, SOX5, SP2, SP7, SPEN, SS18, STAT1, STAT2, STAT3, STAT5A, STRAP, TAF3 (includes EG:209361), TAF6L, TARDBP, TBL1XR1, TBX2, TCEB3, THRAP3, THRB, TLE4, TLR3, TLR4, TOP2A, TRIP11, UBE3A, VDR, WNT5A, YEATS2, ZBTB16, ZHX3, ZNF143, ZNF292, ZNF593

ACVR2A, AFAP1L2, AKAP13, ALOXE3, AR, ARNT, ATF6, BTRC, CAMK1D, CCND2, CCNE1, CD28, CD80 (includes EG:12519), CDCA4, CITED2, CRTC2, CYTIP, DDX58, DPP4, DTX4, EGF (includes EG:13645), EGR1, ETS2, FRK, FUS, FZD2, GABPB1, GADD45G, GLRX3, GNA13, GTF2A1, HBP1, HGF, HNF1A, ID1, ID3 (includes EG:15903), transactivation 6.79E-07 93 IL1A, ILF3, ITSN1, JUN, JUNB, KAT2A, KHDRBS1, KLF6, Macf1, MAF, MED14, MX1, MYCBP2, NCOR1, NDN, NFE2L2, NOTCH4, NR2C2, NR2C2AP, NR3C2 (includes EG:110784), NR4A2, NRIP1, PARP1, PDE8A, PIK3CA, POU2AF1, POU2F1, POU6F1, PPARGC1A, PPP3CA, PRKCZ, PRKDC, PSMC2, PTTG1, PYCARD, RAF1, RARB, RBM39, RBX1, RGS18, RORA, RXRG, SF1, STAT1, STAT2, STAT3, STAT5A, SYK, TBL1XR1, THRB, TRRAP, UAP1, VAV1, VAV2, VDR, WRN, ZBTB16

methylation of DNA 4.03E-04 13 ARID4B, ASZ1, ATF7IP, ATRX, CBX1, CDKN1A, GCM1, GCM2, HELLS, IGF2R, PRMT7, TRDMT1, XIST

AR, ARNT, BTRC, CD28, CD40LG, CDKN1A, CISH, CYP2B6, CYSLTR2, EBF1, EGF (includes EG:13645), EGR1, EPC1, ETS2, FOXP2, FOXP4, GABPB1, GFPT1, GSK3B, GTF2A1, HGF, HIPK2, HIVEP2, HOXA10, HSPA1A/HSPA1B, ID1, ID3 (includes EG:15903), IGFBP1 (includes EG:16006), IL1A, IL1RN, IP6K2, JUN, JUNB, LCP2, LMNA, binding of DNA 5.55E-04 74 MPG, MSX1, NBN, Ncl, NCOR1, NFE2L2, NFIC, NPDC1, NR2C1, NR2C2, NR2C2AP, NR2F2, NR3C2 (includes EG:110784), PAK1, PARP1, PLAU, PLK3, POU2F1, PPARGC1A, PRKCE, PRKDC, PTPN5, PTTG1, RAD21, RAF1, RAPGEF3, RARB, RGCC, RNF17, SART3, STAT1, STAT3, THRB, TRIB3, TSHR, TXLNG, VAV1, VCAM1, VDR

binding of AP1 consensus site 1.42E-03 12 AR, CYSLTR2, EGF (includes EG:13645), HGF, IL1A, JUN, LMNA, NR2F2, PARP1, PLK3, RARB, TSHR AR, ARID4B, CBX2, COPS2, GNAT1, HDAC9, HOXA10, JARID2, JUN, KDM5B, KLF9, MSX1, NCOR1, NR2F2, NR4A2, NRIP1, PAK1, PAX9, RBBP8, RCOR1, SALL1, SIN3A, repression of RNA 1.99E-03 29 SPEN, STAT3, STAT5A, THRB, XIST, ZBTB16, ZNF440/ZNF808 Cell Death 8.56E-08 to 7.30E-03 427 Cell Morphology 1.52E-07 to 5.59E-03 308 Cellular Assembly and Organization 1.84E-07 to 6.24E-03 232 Cellular Function and Maintenance 1.84E-07 to 5.64E-03 358 Page 37 of 42 Diabetes

Supplementary Figure 1. Abnormal blood glucose levels and hypothalamic orexin

expression in diabetic db/db mice. A: Daily changes in blood glucose levels in male db/db

mice at 8 weeks old under ad lib fed condition. n=6. aP<0.05 vs. glucose levels at ZT 5. ZT,

Zeitgeber time. B: Preproorexin mRNA levels in the hypothalamus of male C57BL/6J and

db/db mice at 10 weeks old under ad lib fed condition. n=4-5. The brain tissues were

dissected at ZT 11, and subjected to RT-PCR analysis. **P<0.01. A.U.: arbitrary unit. Data

are the means ± S.E.M.

Supplementary Figure 2. Influences of ICV-orexin A on several pathways controlling

gluconeogenic activity and insulin sensitivity in the liver of mice. A-C: Effects of ICV

administration of orexin A (OXA, 3 nmol, once a day for 3 days) or saline at ZT 14 on

phosphorylation levels of CREB (A), JNK (B), and mTOR (C) in the liver of diabetic db/db

and non-diabetic m+/m+ mice at 20 weeks old. Liver was isolated at ZT 8 (under 6-h fasting

condition) after the last administration. n=3-7. D: Acute effect of ICV-orexin A (3 nmol) on

phosphorylation levels of STAT3 in the liver of 15-week-old db/db mice fasted overnight.

Liver was isolated 120 min after the ICV administration. n=3. Data are the means ± S.E.M.

*P<0.05 and **P<0.01. A.U.: arbitrary unit.

Supplementary Figure 3. Daily nighttime administration of orexin A did not affect daytime

insulin sensitivity in the skeletal muscle of mice. A: Male db/db mice at 10 weeks old were

implanted with ICV cannula and after 7 days of recovery, orexin A (0.3 nmol) or saline was

injected at ZT 14 for 3 days. B: Male C57BL/6J mice at 7-8 weeks old were implanted with

ICV cannula, and then either sham-operated (Sham) or hepatic parasympathectomized (HPx).

After 7-day recovery period, orexin A (3 nmol) or saline was injected at ZT 14 for 3 days. In

the experiments of both A and B, insulin (4 Unit/kg) was intraperitoneally injected at ZT 6 of Diabetes Page 38 of 42

day 3 (under 16-h fasting conditions). Fifteen minutes later, muscle was isolated and subjected to Western blotting to measure the insulin-induced phosphorylation of Akt at Ser473. n=3-6. Data are the means ± S.E.M. *P<0.05 and **P<0.01. A.U., arbitrary unit.

Supplementary Figure 4. Age-related changes in the temporal expression of Per1 and Per2, core clock components, in the liver of Orexin-/- mice under ad lib fed condition. A-D: Liver tissues were isolated from wild-type mice (WT, open circle) and Orexin-/- mice (closed circle) at 3 months old (A and B) and 6 months old (C and D). The mRNA levels of Per1 (A and C) and Per2 (B and D) in the liver were analyzed by real-time RT-PCR. The expression levels were calculated as a ratio to those in WT mice at ZT 8. Data are the means ± S.E.M. n = 3-6 for each time point. *P<0.05 and †P<0.1. ZT, Zeitgeber time.

Page 39 of 42 Diabetes

A 400 db/db a a 300

200

100 Glucose (mg/dL)

0 2 5 8 11 14 17 20 ZT

B Preproorexin 2 **

1 mRNA levels (A.U.) levels mRNA 0 C57BL/6J db/db

Supplementary Fig. 1 Diabetes Page 40 of 42

A B m+/m+ db/db m+/m+ db/db OXA - + - + OXA - + - + p-CREB p-JNK CREB JNK 2 ** ** 2 ***

1 1 (A.U.) (A.U.) p-JNK/JNK p-CREB/CREB 0 0 OXA - + - + OXA - + - + m+/m+ db/db m+/m+ db/db

CD m+/m+ db/db db/db OXA - + - + OXA - +

p-mTOR p-STAT3 mTOR STAT3 ** ** 2 * 2 **

1 1 (A.U.) (A.U.) p-mTOR/mTOR 0 p-STAT3/STAT3 0 OXA - + - + OXA - + m+/m+ db/db

Supplementary Fig. 2 Page 41 of 42 Diabetes

A db/db Saline OXA Insulin - + - +

p-Akt

Akt ** ** 3 2

(A.U.) 1 p-Akt/Akt 0 Insulin - + - + Saline OXA

B Sham HPx Saline OXA Saline OXA Insulin - + - + - + - + p-Akt

Akt

3 * ** * **

2

(A.U.) 1 p-Akt/Akt 0 Insulin - + - + - + - + Saline OXA Saline OXA Sham HPx

Supplementary Fig. 3 Diabetes Page 42 of 42

3 months old 6 months old A C † WT 2 2 Orexin-/-

1 1 mRNA (fold) mRNA mRNA (fold) mRNA

0 0 Per1 Per1 2 8 14 20 2 8 14 20 B D 3 3 2 2 *

mRNA (fold) mRNA 1 (fold) mRNA 1

Per2 0 Per2 0 2 8 14 20 2 8 14 20 ZT ZT

Supplementary Fig. 4