Difference in obesity phenotype between orexin-knockout mice and orexin neuron-deficient mice with same genetic background and environmental conditions

Orexins are a pair of neuropeptides expressed by a population of neurons located in the lateral hypothalamic area (LHA). Prepro-orexin- or orexin receptor type 2-deficient animals exhibit a phenotype remarkably similar to the human sleep disorder, narcolepsy, which is characterized by sleep/wakefulness fragmentation. Human narcolepsy is known to be associated with metabolic abnormalities, including an increased frequency of obesity and non-insulin-dependent diabetes mellitus. Complex disruption of energy homeostasis in orexin neuron-deficient transgenic mice (orexin/ataxin-3 mice) is also manifested as late-onset obesity despite eating less. Here, we report that the development of obesity in orexin neuron-ablated narcoleptic mice is critically dependent on their genetic background and environmental factors, and the phenotype is different from that of prepro-orexin knockout mice even under the same genetic background and environmental factors, suggesting that factors that co-localize in orexin neurons might have important roles in the regulation of energy homeostasis. Our observation also suggests that the obesity observed in orexin neuron-deficient narcolepsy is dependent on the genetic background and environmental factors.     

Hypocretin (orexin) loss in Alzheimer's disease

Sleep disturbances in Alzheimer's disease (AD) patients are associated with the severity of dementia and are often the primary reason for institutionalization. These sleep problems partly resemble core symptoms of narcolepsy, a sleep disorder caused by a general loss of the neurotransmitter hypocretin. AD is a neurodegenerative disorder targeting different brain areas and types of neurons. In this study, we assessed whether the neurodegenerative process of AD also affects hypothalamic hypocretin/orexin neurons. The total number of hypocretin-1 immunoreactive neurons was quantified in postmortem hypothalami of AD patients (n = 10) and matched controls (n = 10). In addition, the hypocretin-1 concentration was measured in postmortem ventricular cerebrospinal fluid of 24 AD patients and 25 controls (including the patients and controls in which the hypothalamic cell counts were performed). The number of hypocretin-1 immunoreactive neurons was significantly decreased by 40% in AD patients (median [25th-75th percentiles]); AD 12,935 neurons (9972-19,051); controls 21,002 neurons (16,439-25,765); p = 0.049). Lower cerebrospinal fluid (CSF) hypocretin-1 levels were found in AD patients compared with controls (AD: 275 pg/mL [197-317]; controls: 320 pg/mL [262-363]; p = 0.038). Two AD patients with documented excessive daytime sleepiness showed the lowest CSF hypocretin-1 concentrations (55 pg/mL and 76 pg/mL). We conclude that the hypocretin system is affected in advanced AD. This is reflected in a 40% decreased cell number, and 14% lower CSF hypocretin-1 levels.     

Role of orexin/hypocretin in reward-seeking and addiction: Implications for obesity

Orexins (also named hypocretins) are recently discovered neuropeptides made exclusively in the hypothalamus. Recent studies have shown that orexin cells located specifically in lateral hypothalamus (LH) are involved in motivated behavior for drugs of abuse as well as natural rewards. Administration of orexin has been shown to stimulate food consumption, and orexin signaling in VTA has been implicated in intake of high-fat food. In self-administration studies, the orexin 1 receptor antagonist SB-334867 (SB) attenuated operant responding for high-fat pellets, sucrose pellets and ethanol, but not cocaine, demonstrating that signaling at orexin receptors is necessary for reinforcement of specific rewards. The orexin system is also implicated in associations between rewards and relevant stimuli. For example, Fos expression in LH orexin neurons varied in proportion to conditioned place preference (CPP) for food, morphine, or cocaine. This Fos expression was altered accordingly for CPP administered during protracted abstinence from morphine or cocaine, when preference for natural rewards was decreased and drug preference was increased. Additionally, orexin has been shown to be involved in reward-stimulus associations in the self-administration paradigm, where SB attenuated cue-induced reinstatement of extinguished sucrose- or cocaine-seeking. Although the specific circuitry mediating the effects of orexin on food reward remains unknown, VTA seems likely to be a critical target for at least some of these orexin actions. Thus, recent studies have established a role for orexin in reward-based feeding, and further investigation is warranted for determining whether function/dysfunction of the orexin system may contribute to the overeating associated with obesity.The paper represents an invited review by a symposium, award winner or keynote speaker at the Society for the Study of Ingestive Behavior [SSIB] Annual Meeting in Portland, July 2009.     

Integration of feeding and spontaneous physical activity: role for orexin

Spontaneous physical activity is activity that is non-volitional, or subconscious, such as fidgeting and shifting in one's seat, and time spent moving (standing and ambulating). Recent evidence indicates that spontaneous physical activity, and the resulting thermogenesis (non-exercise activity thermogenesis) may be regulated by brain systems. A large number of brain areas, with their associated neurotransmitter populations and connectivity, participate in the regulation of feeding behavior by acting as energy sensing and modulating centers. Although less well characterized, it is likely that a multitude of neurotransmitters and brain areas act to mediate spontaneous physical activity. These two behaviors, feeding and spontaneous physical activity, affect energy intake and expenditure and thus are important to body weight. Interestingly, often the two behaviors are affected simultaneously; when feeding is affected, so too is spontaneous physical activity, and both food intake and physical activity (whether spontaneous or volitional) influence activity of brain areas important to both. Several brain areas and neuropeptides are important to feeding and spontaneous physical activity. The lateral hypothalamus is one area that appears important to both behaviors, as stimulation or lesion of this region produces alterations in feeding behavior and spontaneous physical activity. Orexin neurons, with their central location in the lateral hypothalamus, widespread projections and connectivity to other brain areas important to energy homeostasis, are well situated to perform an integrative function. This review focuses on how hypothalamic orexins participate in both feeding and spontaneous physical activity, and provides potential models for the integration of signals important to both.     

Age- and gender-specific changes of hypocretin immunopositive neurons in C57Bl/6 mice

Loss of neurons or neuronal functions over time has been hypothesized to contribute to the dysregulation of autonomic functions observed in aging. In this study, we evaluated the total number of the hypothalamic hypocretin (orexin) immunopositive neurons in 100, 400, 800 and 1000-day-old male and female C57Bl/6 mice that are commonly used in aging studies in vertebrates. Males had 15–20% more hypocretin immunopositive neurons (HIN) than females at all ages examined. Neuronal number for both sexes was stable in the first 400 days of life, but started declining between 400 and 800 days with rates of approximately 1 neuron/day. The rate of loss doubled in males between 800 and 1000 days of age. The total average number of HIN for males was 2251 ± 139 at 100 days, 2235 ± 112 at 400 days, 1914 ± 81 at 800 days, and 1596 ± 301 at 1000 days. The total average number of HIN for females was 1805 ± 76 at 100 days, 1887 ± 118 at 400 days, and 1521 ± 181 at 800 days. Evaluation of the time-dependent decline in the number of hypocretin immunopositive neurons may help to explain the physiological changes in sleep or energy homeostasis regulation during aging.     

营养性肥胖大鼠的脂代谢紊乱与下丘脑中增食欲素A的表达分析

目的: 研究营养性肥胖大鼠下丘脑中增食欲素(orexin A)表达与脂代谢紊乱的规律。 方法: 高脂饮食诱导并评估营养性肥胖大鼠动物模型;采用化学发光免疫分析法和生化酶法测定大鼠血清胰岛素(Ins)、甘油三酯(TG)和总胆固醇(TC);应用实时定量PCR检测下丘脑组织中orexin A mRNA的表达规律。 结果: 大鼠高脂膳食饲养8周后,营养性肥胖大鼠的体重、体脂含量和Lee's指数均高于对照组,血清Ins增加约50％,TG和TC分别增加约94％和43％(P<0.05);营养性肥胖大鼠下丘脑中orexin A的mRNA表达减少约57 % (P<0.05), 且orexin A的表达量与Lee's指数、Ins、TG、TC呈显著负相关,相关系数分别为r=-0.798 (P<0.05)、r=-0.868(P<0.05)、r=-0.981(P<0.05)和r=-0.815(P<0.05)。结论: 在大鼠高脂膳食诱导的营养性肥胖过程中,下丘脑orexin A的低表达和脂代谢紊乱均与营养性肥胖的发生密切相关。     

Localization of the orexin system in the gastrointestinal tract of fallow deer

The aim of the present study was to investigate by immunohistochemistry the presence and distribution of the orexin system in the stomach and gut of fallow deer. Abundant orexin A-positive cells were localized in the middle and basal portions of the mucosal glands of the cardial and fundic regions of the stomach. In the same gastric areas, orexin B-positive cells were also found, mainly localized in the basal portion of glands. In the intestinal tract, orexin-containing cells were occasionally found in the duodenal epithelium and in the rectal intestinal glands. Immunoreactivity for orexin receptors, type 1 and 2 (OX1R and OX2R), was not detected in the same stomach regions. OX1R-immunopositivity was observed in the enteric neuron ganglia localized in the submucosal and muscular intestinal layers, while OX2R-immunopositivity was found close in contact with the cytoplasmic membrane of epithelial cells in the small intestine.     

Sleep is increased in mice with obesity induced by high-fat food

Excessive daytime sleepiness has been associated with obesity in humans. However, experimental studies on sleep in obese animals are scarce and the results are not consistent. To test the hypothesis that obesity is associated with increased sleep, we examined the effects of obesity, induced by high-fat food, on sleep in mice. We first determined baseline sleep in adult C57BL/6 mice (6 months of age). In the following 6 weeks, the experimental mice (n = 12) were switched to high-fat food, in which fat provided 59% of calories, and the control mice (n = 11) were continuously fed with regular lab chows, in which fat provided 16% of calories. The body weights increased steadily in the high-fat group, but maintained constant in the controls. Wakefulness was reduced when assessed after 2, 4, and 6 weeks of high-fat feeding. Concurrently, there were large increases (about 80-100 min/day) in non-rapid eye movement sleep (NREMS). Rapid eye movement sleep (REMS) was not altered. The numbers of NREMS and REMS episodes were increased, whereas the duration of waking episodes was reduced, mainly during the dark period. These alterations in sleep were not observed in the controls. In the high-fat group, the increases of body weight, but not the amounts of energy intake, were negatively correlated with the change in the amounts of wakefulness and positively correlated with the change in the amounts of NREMS. These results indicate that the obese animals have increased sleep pressure and difficulties in maintaining wakefulness during the active phase.     

The wake-promoting effects of hypocretin-1 are attenuated in old rats

Disruption of sleep is a frequent complaint among elderly humans and is also evident in aged laboratory rodents. The neurobiological bases of age-related sleep/wake disruption are unknown. Given the critical role of the hypocretins in sleep/wake regulation, we sought to determine whether the wake-promoting effect of hypocretin changes with age in Wistar rats, a strain in which age-related changes in both sleep and hypocretin signaling have been reported. Intracerebroventricular infusions of hypocretin-1 (10 and 30 μg) significantly increased wake time relative to vehicle in both young (3 mos) and old (25 mos) Wistar rats. However, the magnitude and duration of the wake-promoting effects were attenuated with age. An increase of parameters associated with homeostatic sleep recovery after sleep deprivation, including non-rapid eye movement (NR) sleep time, NR delta power, the ratio of NR to rapid eye movement (REM) sleep, and NR consolidation, occurred subsequent to Hcrt-induced waking in young but not old rats. ICV infusions of hypocretin-2 (10 and 30 μg) produced fewer effects in both young and old rats. These data demonstrate that activation of a major sleep/wake regulatory pathway is attenuated in old rats.     

Respiratory sinus arrhythmia and diseases of aging: obesity, diabetes mellitus, and hypertension

Associations between respiratory sinus arrhythmia (RSA) and several chronic diseases, including obesity, diabetes mellitus, and hypertension, have been documented in recent years. Although most evidence suggests reduced RSA is the result of chronic disease rather than the cause, some studies have documented reduced RSA among at-risk individuals prior to disease onset. These results raise the possibility that decreased vagal tone may play a role in the pathogenesis of certain chronic diseases. Presented here is a brief overview of studies which examine the relationship between vagal tone, as measured by RSA and baroreflex gain, and diseases of aging, including obesity, diabetes mellitus, and hypertension. Mechanisms by which vagal tone may be related to disease processes are discussed. In addition, we present results from a population-based study of RSA and hypertension in older adults. Consistent with previous studies, we found an inverse relationship between RSA and age, cigarette use, and diabetes. In logistic regression models which control for age, cigarette use, and diabetes, we found RSA was a significant negative predictor of hypertension. We conclude that the relationship between RSA and hypertension is somewhat independent of the age-related decline in parasympathetic activity.     

Short sleep is a questionable risk factor for obesity and related disorders: statistical versus clinical significance

Habitually insufficient sleep could contribute towards obesity, metabolic syndrome, etc., via sleepiness-related inactivity and excess energy intake; more controversially, through more direct physiological changes. Epidemiological studies in adult/children point to small clinical risk only in very short (around 5h in adults), or long sleepers, developing over many years, involving hundreds of hours of 'too little' or 'too much' sleep. Although acute 4h/day sleep restriction leads to glucose intolerance and incipient metabolic syndrome, this is too little sleep and cannot be sustained beyond a few days. Few obese adults/children are short sleepers, and few short sleeping adults/children are obese or suffer obesity-related disorders. For adults, about 7h uninterrupted daily sleep is 'healthy'. Extending sleep, even with hypnotics, to lose weight, may take years, compared with the rapidity of utilising extra sleep time to exercise and evaluate one's diet. The real health risk of inadequate sleep comes from a sleepiness-related accident.     

Sleep fragmentation in the elderly: Relationship to daytime sleep tendency

—Sleep in the elderly is known to be disturbed, and many elderly persons also complain of daytime sleepiness. The present study assessed sleep and waking behavior in 12 male (aged 63 to 86) and 12 female (ages 63 to 82) subjects. Sleep stages, respiration, and movement were recorded at night, and daytime sleep tendency was measured using the Multiple Sleep Latency Test during a single 24-hour period. Daytime sleepiness did not correlate with total sleep time or any sleep stage, but was significantly correlated with measures of sleep fragmentation. The latter included transient arousals, a measure of < 15-sec awakenings, and sleep-related respiration disturbance. These findings suggest that fragmented nocturnal sleep is a significant cause of reduced daytime w well-being in elderly individuals. The continuity of both sleep and wakefulness appears to be disrupted with age. Experimental strategies for achieving a rational sleep hygiene are discussed.     

Early life programming of obesity and metabolic disease

It is becoming increasingly apparent that conditions experienced in early life play an important role in the long-term health of individuals. Alterations in development due to impaired, excessive or imbalanced growth, both in utero and during critical periods of relative plasticity beyond birth, can lead to the permanent programming of physiological systems. The regulation of energy balance is one area that is receiving particular attention, as rates of obesity and associated metabolic and cardiovascular disease continue to rise. Over recent decades, much progress has been made toward understanding the way in which metabolic tissues and physiological systems develop, and the impact of early life events and nutrition on these processes. It is apparent within human populations that some individuals are better able to maintain an appropriate body weight in the face of an obesogenic environment. Animal models have been widely used for the investigation of differential susceptibility to diet-induced obesity (DIO) and impaired energy balance regulation, and are shedding light on key pathways that may be involved. Alterations in pathways mediating energy homeostasis, outlined below, are likely candidates for programming effects following disturbed growth in early life.     

Sleep fragmentation in the elderly: Relationship to daytime sleep tendency

—Sleep in the elderly is known to be disturbed, and many elderly persons also complain of daytime sleepiness. The present study assessed sleep and waking behavior in 12 male (aged 63 to 86) and 12 female (ages 63 to 82) subjects. Sleep stages, respiration, and movement were recorded at night, and daytime sleep tendency was measured using the Multiple Sleep Latency Test during a single 24-hour period. Daytime sleepiness did not correlate with total sleep time or any sleep stage, but was significantly correlated with measures of sleep fragmentation. The latter included transient arousals, a measure of < 15-sec awakenings, and sleep-related respiration disturbance. These findings suggest that fragmented nocturnal sleep is a significant cause of reduced daytime w well-being in elderly individuals. The continuity of both sleep and wakefulness appears to be disrupted with age. Experimental strategies for achieving a rational sleep hygiene are discussed.     

Chronobiology of aging: Temperature, sleep-wake rhythms and entrainment

—Studies were carried out on a group of six young (ages 23–30) and six older (ages 53–70) normal men who lived under conditions of temporal, but not social isolation, from three to eight weeks. During entrained and non-entrained (free-running) conditions, comparative measurements were made of sleep-wake cycles, sleep stages and rectal temperature rhythms for these two age groups. Results demonstrated a reduction in the period and amplitude of the body temperature rhythms during free-running in the older group. Sleep efficiency, total sleep time, REM sleep latency, REM episode length, percent REM in the last 2 hours of sleep, the length and frequency of arousals during sleep, and the terminal wake latency were all age related and dependent on entrainment. The period of the sleep-wake cycle, terminal awakenings from REM and percent REM in the first 3 hours of sleep were not age related but were dependent on entrainment. Sleep stages as percents of total sleep time were found to be age related but independent of entrainment, while sleep latency, mid-REM to mid-REM cycle length and the ratio of sleep to total time were neither age related nor dependent on entrainment. In addition, individual chronobiological differences were prominent in the older group. Changes of period and of the phase relationship of sleep-wake and temperature rhythms occurred in several subjects during the non-entrained condition.     

Chronobiology of aging: Temperature, sleep-wake rhythms and entrainment

—Studies were carried out on a group of six young (ages 23–30) and six older (ages 53–70) normal men who lived under conditions of temporal, but not social isolation, from three to eight weeks. During entrained and non-entrained (free-running) conditions, comparative measurements were made of sleep-wake cycles, sleep stages and rectal temperature rhythms for these two age groups. Results demonstrated a reduction in the period and amplitude of the body temperature rhythms during free-running in the older group. Sleep efficiency, total sleep time, REM sleep latency, REM episode length, percent REM in the last 2 hours of sleep, the length and frequency of arousals during sleep, and the terminal wake latency were all age related and dependent on entrainment. The period of the sleep-wake cycle, terminal awakenings from REM and percent REM in the first 3 hours of sleep were not age related but were dependent on entrainment. Sleep stages as percents of total sleep time were found to be age related but independent of entrainment, while sleep latency, mid-REM to mid-REM cycle length and the ratio of sleep to total time were neither age related nor dependent on entrainment. In addition, individual chronobiological differences were prominent in the older group. Changes of period and of the phase relationship of sleep-wake and temperature rhythms occurred in several subjects during the non-entrained condition.     

Developmental and aging change of orexin-A and -B immunoreactive neurons in the male rat hypothalamus

Orexin/hypocretin is indicated to affect various physiological functions and behaviors, such as energy balance, feeding, wake–sleep cycle, stress response, and reproduction. This study investigated postnatal development and aging changes of the orexin neuron in the male rat hypothalamus. The brain tissue of rats from 1 week to 24 months old was analyzed by immunohistochemistry for two forms of orexin peptides, orexin-A and -B. The number of immunoreactive cells for each age group was counted and the immunoreactive intensity was also analyzed in order to reveal the changes in the number of expressing cells and the relative amount of the peptides. The number of orexin immunoreactive cells increased from postnatal 2 weeks to maturation, then slightly decreased and stabilized until the age of 8 months old, but it was significantly decreased by 24 months old. The intensity of the immunoreaction followed almost the same pattern. Our findings demonstrate that orexin neurons are increased during maturation and then are significantly decreased during the period from 8 to 24 months old, indicating an involvement of orexin in the physiological changes in rat aging such as energy balance, sleep, stress response, and reproduction.     

Immunohistochemical localization of orexin A and orexin type 2 receptor-positive cells in the placenta of dogs

The aim of the present study was to examine the presence and distribution of cells that express immunopositivity for orexin A (OXA) and its type 2 receptor (OX2R) in the dog placenta toward the end of pregnancy using immunohistochemical techniques. In the placental fetal portion, a few OXA and OX2R-positive cells were seen scattered in the outermost coating layer of chorionic villi and in the trophoblastic protrusions. Closer to the maternal portion, immunopositive labeling for both peptides was visible in the glandular epithelia and that for OXA also in the endothelium of the capillaries. These observations allow us to hypothesize that the canine placenta may be not only a source of orexin A, but also its target, and that orexin A may play an important role in controlling the function of this important organ for normal fetal development.     

Sleep related breathing disorders in older men: A search for underlying mechanisms

—The incidence of sleep-related breathing disorders (SRBDs) associated with hemoglobin desaturation was determined by nocturnal polygraphic evaluations in 26 healthy men, aged 55–70 years. Sixteen subjects (62%) had abnormal rates of at least 12 episodes per hour of sleep: 8 had occlusive, and 8 had central apnea or hypopnea. During waking ten of 16 SRBD subjects and only one subject without SRBDs exhibited either an elevated nasopharyngeal airway resistance (n=4) or a reduced ventilatory response to hypercapnia (n=4) and/or hypoxia (n=3). However, these abnormalities were not related to the type or severity of SRBDs, and 6 subjects with SRBDs demonstrated no respiratory defect. We conclude that SRBDs have a very high incidence in older males and are not usually secondary to pulmonary cardiac, neurological, or behavioral disorders. Additionally, we hypothesize that abnormalities in ventilatory control or upper airway resistance contribute to SRBDs, but depression of brain stem reticular formation activity during sleep plays a primary role in these disorders. Factors related to both aging and SRBDs are reviewed. These include reduced chemoreceptor responses, altered steroid hormone metabolism, and use and metabolism of hypnotic drugs and alcohol.