Social stress alters sleep in FGF21-deficient mice.

Although several previous studies have suggested a relationship between sleep and the stress response, the mechanism underlying this relationship remains largely unknown. Here, we show that fibroblast growth factor 21 (FGF21), a lipid metabolism-related hormone, may play a role in this relationship. In this study, we examined differences in the stress response between FGF21 knockout (KO) mice and wild-type (WT) mice after social defeat stress (SDS). When the amount of non-rapid eye movement (NREM) sleep, rapid eye movement (REM) sleep and wakefulness were averaged over the dark period after SDS, only KO mice showed significant differences in NREM sleep and wakefulness. In the social interaction test, KO mice seemed to be more prone to social avoidance. Our real-time (RT) -PCR results revealed that the mRNA expression of the stress- and sleep-related gene gamma-aminobutyric acid A receptor subunit alpha 2 was significantly lower in WT mice than in KO mice. Moreover, KO mice showed lower plasma levels of ketone bodies, which also affect sleep/wake regulation, than WT mice. These results suggested that FGF21 might influence sleep/wake regulation by inducing production of an anti-stress agent and/or ketone bodies, which may result in resilience to social stress.

Dual orexin receptor antagonist drug suvorexant can help in amelioration of predictable chronic mild stress-induced hyperalgesia.

AIMS: This study aimed to evaluate the involvement of the orexin system in predictable chronic mild stress (PCMS) and the effects of suvorexant, a dual orexin receptor antagonist, on nociceptive behavior in PCMS. MATERIALS AND METHODS: Male C57BL/6 J mice were separated into various PCMS groups: a control group with sawdust on the floor of the rearing cage (C), a group with mesh wire on the floor (M), and a group with water just below the mesh wire (W). Activation of lateral hypothalamic orexin neurons was assessed using immunofluorescence. In another experiment, half of the mice in each group were administered an intraperitoneal injection of suvorexant (10 mg/kg), and the remaining mice were injected with the same amount of vehicle (normal saline). Thermal hyperalgesia was examined using tail immersion and hot plate tests, while mechanical hyperalgesia was investigated using the tail pinch test after 21 days of PCMS. KEY FINDINGS: Animals subjected to PCMS showed an increased percentage of activated orexin neurons in the lateral hypothalamic region after 21 days. Mice raised in the PCMS environment showed increased pain sensitivity in several pain tests; however, the symptoms were significantly reduced by suvorexant administration. SIGNIFICANCE: The findings revealed that PCMS activates hypothalamic orexin neuronal activity, and the use of suvorexant can help attenuate PCMS-induced thermal and mechanical hyperalgesia.

Short-term physical inactivity induces diacylglycerol accumulation and insulin resistance in muscle via lipin1 activation.

Physical inactivity impairs muscle insulin sensitivity. However, its mechanism is unclear. To model physical inactivity, we applied 24-h hind-limb cast immobilization (HCI) to mice with normal or high-fat diet (HFD) and evaluated intramyocellular lipids and the insulin signaling pathway in the soleus muscle. Although 2-wk HFD alone did not alter intramyocellular diacylglycerol (IMDG) accumulation, HCI alone increased it by 1.9-fold and HCI after HFD further increased it by 3.3-fold. Parallel to this, we found increased protein kinase C ε (PKCε) activity, reduced insulin-induced 2-deoxyglucose (2-DOG) uptake, and reduced phosphorylation of insulin receptor ß (IRß) and Akt, key molecules for insulin signaling pathway. Lipin1, which converts phosphatidic acid to diacylglycerol, showed increase of its activity by HCI, and dominant-negative lipin1 expression in muscle prevented HCI-induced IMDG accumulation and impaired insulin-induced 2-DOG uptake. Furthermore, 24-h leg cast immobilization in human increased lipin1 expression. Thus, even short-term immobilization increases IMDG and impairs insulin sensitivity in muscle via enhanced lipin1 activity.NEW & NOTEWORTHY Physical inactivity impairs muscle insulin sensitivity. However, its mechanism is unclear. To model physical inactivity, we applied 24-h hind-limb cast immobilization to mice with normal or high-fat diet and evaluated intramyocellular lipids and the insulin signaling pathway in the soleus muscle. We found that even short-term immobilization increases intramyocellular diacylglycerol and impairs insulin sensitivity in muscle via enhanced lipin1 activity.

Feeding Rhythm-Induced Hypothalamic Agouti-Related Protein Elevation via Glucocorticoids Leads to Insulin Resistance in Skeletal Muscle.

Circadian phase shifts in peripheral clocks induced by changes in feeding rhythm often result in insulin resistance. However, whether the hypothalamic control system for energy metabolism is involved in the feeding rhythm-related development of insulin resistance is unknown. Here, we show the physiological significance and mechanism of the involvement of the agouti-related protein (AgRP) in evening feeding-associated alterations in insulin sensitivity. Evening feeding during the active dark period increased hypothalamic AgRP expression and skeletal muscle insulin resistance in mice. Inhibiting AgRP expression by administering an antisense oligo or a glucocorticoid receptor antagonist mitigated these effects. AgRP-producing neuron-specific glucocorticoid receptor-knockout (AgRP-GR-KO) mice had normal skeletal muscle insulin sensitivity even under evening feeding schedules. Hepatic vagotomy enhanced AgRP expression in the hypothalamus even during ad-lib feeding in wild-type mice but not in AgRP-GR-KO mice. The findings of this study indicate that feeding in the late active period may affect hypothalamic AgRP expression via glucocorticoids and induce skeletal muscle insulin resistance.

Pain sensitivity increases with sleep disturbance under predictable chronic mild stress in mice.

Even though it has been well documented that stress can lead to the development of sleep disorders and the intensification of pain, their relationships have not been fully understood. The present study was aimed at investigating the effects of predictable chronic mild stress (PCMS) on sleep-wake states and pain threshold, using the PCMS rearing conditions of mesh wire (MW) and water (W) for 21 days. Exposure to PCMS decreased the amount of non-rapid eye movement (NREM) sleep during the dark phase. Moreover, the chronicity of PCMS decreased slow-wave activity (SWA) during NREM sleep in the MW and W groups in both the light and dark phases. Mechanical and aversively hot thermal hyperalgesia were more intensified in the PCMS groups than the control. Higher plasma corticosterone levels were seen in mice subjected to PCMS, whereas TNF-α expression was found higher in the hypothalamus in the W and the trigeminal ganglion in the MW group. The W group had higher expression levels of IL-6 in the thalamus as well. The PCMS paradigm decreased SWA and may have intensified mechanical and thermal hyperalgesia. The current study also suggests that rearing under PCMS may cause impaired sleep quality and heightened pain sensation to painful mechanical and aversively hot thermal stimuli.

Intracranial mast cells contribute to the control of social behavior in male mice.

Mast cells (MCs) exist intracranially and have been reported to affect higher brain functions in rodents. However, the role of MCs in the regulation of emotionality and social behavior is unclear. In the present study, using male mice, we examined the relationship between MCs and social behavior and investigated the underlying mechanisms. Wild-type male mice intraventricularly injected with a degranulator of MCs exhibited a marked increase in a three-chamber sociability test. In addition, removal of MCs in Mast cell-specific Toxin Receptor-mediated Conditional cell Knock out (Mas-TRECK) male mice showed reduced social preference levels in a three-chamber sociability test without other behavioral changes, such as anxiety-like and depression-like behavior. Mas-TRECK male mice also had reduced serotonin content and serotonin receptor expression and increased oxytocin receptor expression in the brain. These results suggested that MCs may contribute to the regulation of social behavior in male mice. This effect may be partially mediated by serotonin derived from MCs in the brain.

Deletion of H-ferritin in macrophages alleviates obesity and diabetes induced by high-fat diet in mice.

AIMS/HYPOTHESIS: Iron accumulation affects obesity and diabetes, both of which are ameliorated by iron reduction. Ferritin, an iron-storage protein, plays a crucial role in iron metabolism. H-ferritin exerts its cytoprotective action by reducing toxicity via its ferroxidase activity. We investigated the role of macrophage H-ferritin in obesity and diabetes. METHODS: Conditional macrophage-specific H-ferritin (Fth, also known as Fth1) knockout (LysM-Cre Fth KO) mice were used and divided into four groups: wild-type (WT) and LysM-Cre Fth KO mice with normal diet (ND), and WT and LysM-Cre Fth KO mice with high-fat diet (HFD). These mice were analysed for characteristics of obesity and diabetes, tissue iron content, inflammation, oxidative stress, insulin sensitivity and metabolic measurements. RAW264.7 macrophage cells were used for in vitro experiments. RESULTS: Iron concentration reduced, and mRNA expression of ferroportin increased, in macrophages from LysM-Cre Fth KO mice. HFD-induced obesity was lower in LysM-Cre Fth KO mice than in WT mice at 12 weeks (body weight: KO 34.6 ± 5.6 g vs WT 40.1 ± 5.2 g). mRNA expression of inflammatory cytokines and infiltrated macrophages and oxidative stress increased in the adipose tissue of HFD-fed WT mice, but was not elevated in HFD-fed LysM-Cre Fth KO mice. However, WT mice fed an HFD had elevated iron concentration in adipose tissue and spleen, which was not observed in LysM-Cre Fth KO mice fed an HFD (adipose tissue [µmol Fe/g protein]: KO 1496 ± 479 vs WT 2316 ± 866; spleen [µmol Fe/g protein]: KO 218 ± 54 vs WT 334 ± 83). Moreover, HFD administration impaired both glucose tolerance and insulin sensitivity in WT mice, which was ameliorated in LysM-Cre Fth KO mice. In addition, energy expenditure, mRNA expression of thermogenic genes, and body temperature were higher in KO mice with HFD than WT mice with HFD. In vitro experiments showed that iron content was reduced, and lipopolysaccharide-induced Tnf-α (also known as Tnf) mRNA upregulation was inhibited in a macrophage cell line transfected with Fth siRNA. CONCLUSIONS/INTERPRETATION: Deletion of macrophage H-ferritin suppresses the inflammatory response by reducing intracellular iron levels, resulting in the prevention of HFD-induced obesity and diabetes. The findings from this study highlight macrophage iron levels as a potential therapeutic target for obesity and diabetes.

Sleep profile during fasting in PPAR-alpha knockout mice.

Peroxisome proliferator-activated receptor alpha (PPARα) is a transcription factor that belongs to the nuclear receptor family and plays an important role in regulating gene expression associated with lipid metabolism. PPARα promotes hepatic fatty acid oxidation and ketogenesis in response to fasting. Because energy metabolism is known to affect sleep regulation, manipulations that change PPARα are likely to affect sleep and other physiological phenotypes. In this study, we examined the role of PPARα in sleep/wake regulation using PPARα knockout (KO) mice. Sleep, body temperature (BT), locomotor activity, arterial pressure (AP) and heart rate (HR) were recorded in KO mice and wild-type (WT) controls under ad libitum-fed conditions and 24-hour food deprivation (FD). KO and WT mice were identical in basal sleep amount, BT, mean AP and HR, although KO mice showed enhanced sleepiness (enhanced EEG slow-wave activity). In response to FD, KO mice showed a large drop in wakefulness and locomotor activity at the end of the dark phase, whereas WT mice did not. Similarly, AP and HR, which were suppressed by FD, decreased more in KO than in WT mice. Compared to WT mice, KO mice showed a reduced concentration of plasma ketone bodies and decreased mRNA expression of the ketogenic enzyme gene Hmgcs2 in the liver and brain under FD conditions. These results suggest that PPARα and/or lipid metabolism is involved in the maintenance of wakefulness and locomotor activity during fasting in mice.

Dopamine stimulation of the septum enhances exercise efficiency during complicated treadmill running in mice.

We aimed to identify the neurotransmitters and brain regions involved in exercise efficiency in mice during continuous complicated exercises. Male C57BL/6J mice practiced treadmill running with intermittent obstacles on a treadmill for 8 days. Oxygen uptake (VO2) during treadmill running was measured as exercise efficiency. After obstacle exercise training, the VO2 measured during treadmill running with obstacles decreased significantly. Obstacle exercise-induced c-Fos expressions and dopamine turnover (DOPAC/dopamine) in the septum after obstacle exercise training were significantly higher than that before training. The dopamine turnover was correlated with exercise efficiency on the 3rd day after exercise training. Furthermore, the training effect on exercise efficiency was significantly decreased by injection of dopamine receptor antagonists into the septum and was associated with decreased c-Fos expressions in the septum and hippocampus of the mice. These results suggest that dopaminergic function in the septum is involved in exercise efficiency during continuous complicated exercises.

Role of orexin in exercise-induced leptin sensitivity in the mediobasal hypothalamus of mice.

Orexin is known as an important neuropeptide in the regulation of energy metabolism. However, the role of orexin in exercise-induced leptin sensitivity in the hypothalamus has been unclear. In this study, we determined the effect of transient treadmill exercise on leptin sensitivity in the mediobasal hypothalamus (MBH) of mice and examined the role of orexin in post-exercise leptin sensitivity. Treadmill running for 45 min increased the orexin neuron activity in mice. Intraperitoneal injection of a submaximal dose of leptin after exercise stimulated the phosphorylation of signal transducer and activator of transcription 3 (STAT3) in MBH of mice post-exercise compared with that in non-exercised mice, although intracerebroventricular (icv) injection of leptin did not enhance STAT3 phosphorylation, even after exercise. Icv injection of an orexin receptor antagonist, SB334867 reduced STAT3 phosphorylation, which was enhanced by icv injection of orexin but not by direct injection of orexin into MBH. Exercise increased the phosphorylation of extracellular signal-regulated kinases (ERKs) in the MBH of mice, while ERK phosphorylation was reduced by SB334867. Leptin injection after exercise increased the leptin level in MBH, whereas icv injection of SB334867 suppressed the increase in the leptin level in MBH of mice. These results indicate that the activation of orexin neurons by exercise may contribute to the enhancement of leptin sensitivity in MBH. This effect may be mediated by increased transportation of circulating leptin into MBH, with the involvement of ERK phosphorylation.

Sufficient intake of high-fat food attenuates stress-induced social avoidance behavior.

AIMS: Psychosocial stress is a form of mental stress associated with human relationships that underlies the pathogenesis of mental disorders such as depression. Previous studies have suggested that intake of energy-dense foods, also known as "palatable foods," can relieve psychosocial stress. However, it remains unclear whether the volume of palatable food affects abnormal behavior induced by psychosocial stress. In the present study, we aimed to determine whether levels of high-fat food intake significantly influence psychosocial stress using the social-defeat stress (SDS) paradigm. MAIN METHODS: Mice subjected to SDS ate either a high-fat or normal chow diet for 10 days. Behavioral tests were conducted following the completion of the SDS paradigm. The hypothalamus, liver, and blood were examined post-mortem. KEY FINDINGS: Mice with sufficient intake of high-fat chow immediately following exposure to SDS did not exhibit social avoidance behavior, suggesting that a high-fat diet may improve social behavior. However, inadequate intake of high-fat food, which did not alter cholesterol metabolism or hypothalamic-pituitary-adrenal axis activity, was not associated with such benefits, instead increased anxiety-like behavior. SIGNIFICANCE: The results of the present study demonstrate that eating a high-fat diet may attenuate stress, but that this benefit disappears with insufficient intake of high-fat foods. The benefits of a high-fat diet under SDS may be related to cholesterol metabolism in the liver.

Enhancement of fear learning in PPARα knockout mice.

Peroxisome proliferator-activated receptor alpha (PPARα) is a member of the nuclear receptor superfamily and regulates fatty acid oxidation. Although PPARα is expressed not only in the peripheral tissues but also in the brain, its role in higher brain function is unclear. In this study, we investigated the role of PPARα in the control of behavior, including memory/learning and mood change, using PPARα knockout (KO) mice. A significant difference between wild-type (WT) and KO mice was seen in the passive avoidance test, demonstrating that KO mice showed enhanced fear leaning. In the amygdala of KO mice, the levels of dopamine and its metabolites were increased, and the mRNA expression of dopamine degrading enzyme was decreased. When dopamine D1 receptor antagonist was administered, the enhanced fear learning observed in KO mice was attenuated. These results suggest that PPARα is involved in the regulation of emotional memory via the dopamine pathway in the amygdala.

[Modification of Energy Metabolism and Higher Brain Function by Feeding Rhythm].

　A molecular clock exists within almost all organizations in a living body, and these clocks influence the periodicity of many physiological phenomena such as eating behaviors, the sleep-wake cycle, and hormone secretion. Especially, a living body's energy metabolism is involved with the molecular clocks genetically driven in peripheral tissues, which act in strong relation to eating rhythms. However, the possibility that rhythms may also have an inverse influence has recently been pointed out. In this manuscript, we review the outline of circadian rhythms, then refer to the possibility that a clock gene in the peripheral tissues, capable of being changed by eating rhythms, may influence sleep-wake regulation and energy metabolism.

Activation of AMPK-Regulated CRH Neurons in the PVH is Sufficient and Necessary to Induce Dietary Preference for Carbohydrate over Fat.

Food selection is essential for metabolic homeostasis and is influenced by nutritional state, food palatability, and social factors such as stress. However, the mechanism responsible for selection between a high-carbohydrate diet (HCD) and a high-fat diet (HFD) remains unknown. Here, we show that activation of a subset of corticotropin-releasing hormone (CRH)-positive neurons in the rostral region of the paraventricular hypothalamus (PVH) induces selection of an HCD over an HFD in mice during refeeding after fasting, resulting in a rapid recovery from the change in ketone metabolism. These neurons manifest activation of AMP-activated protein kinase (AMPK) during food deprivation, and this activation is necessary and sufficient for selection of an HCD over an HFD. Furthermore, this effect is mediated by carnitine palmitoyltransferase 1c (CPT1c). Thus, our results identify the specific neurons and intracellular signaling pathway responsible for regulation of the complex behavior of selection between an HCD and an HFD. VIDEO ABSTRACT.

Induction of glucose uptake in skeletal muscle by central leptin is mediated by muscle ß2-adrenergic receptor but not by AMPK.

Leptin increases glucose uptake and fatty acid oxidation (FAO) in red-type skeletal muscle. However, the mechanism remains unknown. We have investigated the role of ß2-adrenergic receptor (AR), the major ß-AR isoform in skeletal muscle, and AMPK in leptin-induced muscle glucose uptake of mice. Leptin injection into the ventromedial hypothalamus (VMH) increased 2-deoxy-D-glucose (2DG) uptake in red-type skeletal muscle in wild-type (WT) mice accompanied with increased phosphorylation of the insulin receptor (IR) and Akt as well as of norepinephrine (NE) turnover in the muscle. Leptin-induced 2DG uptake was not observed in ß-AR-deficient (ß-less) mice despite that AMPK phosphorylation was increased in the muscle. Forced expression of ß2-AR in the unilateral hind limb of ß-less mice restored leptin-induced glucose uptake and enhancement of insulin signalling in red-type skeletal muscle. Leptin increased 2DG uptake and enhanced insulin signalling in red-type skeletal muscle of mice expressing a dominant negative form of AMPK (DN-AMPK) in skeletal muscle. Thus, leptin increases glucose uptake and enhances insulin signalling in red-type skeletal muscle via activation of sympathetic nerves and ß2-AR in muscle and in a manner independent of muscle AMPK.

Mast cell involvement in glucose tolerance impairment caused by chronic mild stress with sleep disturbance.

We have developed a chronic mild stress (MS) mouse model by simply rearing mice on a wire net for 3 weeks and investigated the effects of MS on glucose homeostasis and sleep. MS mice showed impaired glucose tolerance and disturbed sleep. One-week treatment with a histamine H1 receptor antagonist (H1RA) ameliorated the glucose intolerance and improved sleep quality in MS mice. MS mice showed an increased number of mast cells in both adipose tissue and the brain. Inhibition of mast cell function ameliorated the impairment in both glucose tolerance and sleep. Together, these findings indicate that mast cells may represent an important pathophysiological mediator in sleep and energy homeostasis.

Intracerebroventricular injection of ghrelin decreases wheel running activity in rats.

There is an increasing interest in elucidating the molecular mechanisms by which voluntary exercise is regulated. In this study, we examined how the central nervous system regulates exercise. We used SPORTS rats, which were established in our laboratory as a highly voluntary murine exercise model. SPORTS rats showed lower levels of serum ghrelin compared with those of the parental line of Wistar rats. Intracerebroventricular and intraperitoneal injection of ghrelin decreased wheel-running activity in SPORTS rats. In addition, daily injection of the ghrelin inhibitor JMV3002 into the lateral ventricles of Wistar rats increased wheel-running activity. Co-administration of obestatin inhibited ghrelin-induced increases in food intake but did not inhibit ghrelin-induced suppression of voluntary exercise in rats. Growth hormone secretagogue receptor (GHSR) in the hypothalamus and hippocampus of SPORTS rats was not difference that in control rats. We created an arcuate nucleus destruction model by administering monosodium glutamate (MSG) to neonatal SPORTS rats. Injection of ghrelin into MSG-treated rats decreased voluntary exercise but did not increase food intake, suggesting that wheel-running activity is not controlled by the arcuate nucleus neurons that regulate feeding. These results provide new insights into the mechanism by which ghrelin regulates voluntary activity independent of arcuate nucleus neurons.

Late feeding in the active period decreases slow-wave activity.

AIMS: Sleep and feeding behaviors closely interact to maintain energy homeostasis. While it is known that sleep disorders can lead to various metabolic issues such as insulin resistance, the mechanism for this effect is poorly understood. We thus investigated whether different feeding rhythms during the active period affect sleep-wake regulation. MAIN METHODS: For 2weeks, mice were randomly assigned to 1 of 3 feeding schedules as follows: free access to lab chow during the active period (ZT12-24, Ad-lib group), free access to lab chow during the first half of the active period (ZT12-18; Morning group), or free access to lab chow during the second half of the active period (ZT18-24, Evening group). Food intake, body weight, body temperature, locomotor activity, and sleep were evaluated. The hypothalamus and cerebral cortex were examined post-mortem. KEY FINDINGS: No alterations in food intake or body weight were observed among the 3 groups. The Evening group showed lower slow-wave activity (SWA) than the other 2 groups, in addition to higher expression of orexin mRNA in the hypothalamus and higher concentrations of dopamine and its metabolites in the cerebral cortex. AMPK phosphorylation was increased in the hypothalamus of mice in the Evening group; however, AMPK inhibition had no effect on SWA. SIGNIFICANCE: We concluded that late feeding reduces SWA in NREM sleep via a mechanism that involves orexin-mediated arousal in the hypothalamus and elevated monoamines in the cerebral cortex. These data have important implications for the relationship between sleep-wake disturbances and metabolic disorders.

Intracerebroventricular injection of adiponectin regulates locomotor activity in rats.

Enhancing exercise motivation is the best way to prevent obesity and diabetes. In this study, we examined whether adiponectin affects locomotion activity in Wister and Spontaneously-Running Tokushima-Shikoku (SPORTS) rats using two types of behavioral assays: home cage and wheel running activity. SPORTS rats were established from an original line from Wister strain that had shown high level of wheel running activity in our laboratory. Injection of adiponectin into the lateral ventricle of Wister rats and SPORTS rats decreased home cage activity, but no change was observed in the food intake and oxygen consumption. This result indicates the possibility that adiponectin can reduce non-exercise activity thermogenesis (NEAT) and physical activity via the central nervous system. In contrast, injection of adiponectin did not change wheel running activity in SPORTS rats. We produced hypothalamus-destructed model rat using monosodium glutamate (MSG) to elucidate the regulation site of adiponectin. Injection of adiponectin into MSG-treated SPORTS rats did not change amount of home cage activity and food intake, suggesting that adiponectin action on home cage activity was in the hypothalamic area. These results suggest that adiponectin regulates locomotion activity through mediobasal hypothalamus.

Voluntary exercise and increased food intake after mild chronic stress improve social avoidance behavior in mice.

It is well-established that exercise can influence psychological conditions, cognitive function, and energy metabolism in peripheral tissues including the skeletal muscle. However, it is not clear whether exercise can influence social interaction with others and alleviate defeat stress. This study investigated the effect of voluntary wheel running on impaired social interaction induced by chronic social defeat stress (SDS) using the resident-intruder social defeat model. Mice were divided into three groups: control, stress alone, and stress+exercise. SDS was performed by exposing C57BL/6 mice to retired ICR mice for 2.5 min. The C57BL/6 mice were continuously defeated by these resident (aggressor) mice and, following 5 days of SDS, experienced 2 days of rest with no SDS. Mice in the stress+exercise group were allowed to voluntarily run on a wheel for 2h after every SDS exposure. Two weeks later, compared to the control group, the stress group showed a higher ratio of time spent in the corner zone of a social interaction paradigm even though SDS did not elicit depressive- and anxiety-like behaviors. We also observed that voluntary exercise, which did not affect muscle weight and gene expression, decreased social avoidance behavior of stressed mice without clear changes in brain monoamine levels. Interestingly, food intake in the stress+exercise group was the greatest among the three groups. To test the effect of the exercise-induced increase in food intake on social behavior, we set up a pair-fed group where food intake was restricted. We then compared these mice to mice in the stress alone group. We found that the ratio of time spent in the corner zone of the social interaction test was not different between ad libitum- and pair-fed groups, although pair-fed mice spent more time in the corner zone when an aggressor mouse was present than when it was absent. In addition, pair-feeding did not show exercise-induced reductions of adrenal gland weight and enhanced the loss of body fat. Our findings indicate that voluntary exercise reduces social avoidance behavior induced by SDS. Further, we determined that SDS and exercise-induced increases in food intake partially influence energy metabolism and social avoidance behavior.