Changes in central and peripheral inflammatory responses to lipopolysaccharide in ovariectomized female rats.

Obesity leads to increases in inflammatory responses in a site-specific manner. Ovariectomized animals, usually used as menopause models, exhibit obesity; however, their inflammatory responses have not been fully examined. In the present study, we investigated whether ovariectomy had site-specific effects on inflammatory responses. First, fever and anorectic responses to systemic injections of lipopolysaccharide (LPS) (500µg/kg, i.p.) were compared between ovariectomized rats (OVX) and sham-operated female rats (Sham). Inflammatory cytokines at the central and peripheral levels were also compared under saline-injected and LPS-injected conditions. Body weight in OVX was significantly higher than in Sham. The anorectic responses (reduction of body weight and food intake) to LPS were higher in OVX than in Sham. In the hypothalamus, all of the examined cytokine (IL-1ß, TNF-α and IL-6) mRNA levels in OVX were higher than in Sham under the LPS-injected condition. On the other hand, in serum and adipose tissue, only IL-6, not IL-1ß and TNF-α, levels in OVX were significantly higher than those in Sham under the LPS-injected condition. Second, responses to central (intracerebroventricular) injections of LPS (500ng) were compared between OVX and Sham. The result was that the fever response in OVX was more evident than in Sham. Finally, responses to systemic injections of LPS (500µg/kg, i.p.) were compared between OVX (OVX-oil) and OVX with estradiol (E) and progesterone (P) supplementation (OVX-EP). The anorectic responses and hypothalamic cytokine mRNA levels under LPS-injected condition were not different between OVX-oil and OVX-EP. These results indicate that ovariectomy enhances inflammatory responses, especially at the central level compared with the peripheral level. As supplementation of E and P could not attenuate the anorectic and cytokine responses to LPS, the deficiency of gonadal steroids might not be directly involved in the increase of inflammatory responses in OVX.

Developmental changes in the responsiveness of hypothalamic ER alpha mRNA levels to food deprivation.

OBJECTIVE: Estrogen plays an important role in feeding and energy balance, and the critical role of estrogen in the control of appetite and energy balance is mediated by hypothalamic estrogen receptor (ER) alpha. In undernourished rodents, hypothalamic ER alpha mRNA expression are decreased. Responses of some hypothalamic factors to negative energy balance develop during the early neonatal period. DESIGN: In this study, we examined the developmental changes of fasting-induced alterations in hypothalamic ER alpha mRNA expression in female rats. RESULTS: ER alpha mRNA expression was reduced after a 12-h or 24-h fast at postnatal days 15 and 25, but not at day 5. Serum estradiol levels in postnatal day 25 rats were not changed by fasting. Although serum leptin levels were suppressed by fasting at all ages, hypothalamic ER alpha mRNA expression at postnatal day 25 was not changed by leptin administration after a 24-h fast. CONCLUSIONS: These data show that the sensitivity of hypothalamic ER alpha to negative energy balance may not be established in the early neonatal period, and that it develops by postnatal day 15. Decreased leptin levels might not be involved in the alterations of hypothalamic ER alpha mRNA expression in the undernourished condition.

Prepubertal exposure to glucocorticoid delays puberty independent of the hypothalamic Kiss1-GnRH system in female rats.

Secretion of glucocorticoids is widely known as a key endocrine response to stresses. Prenatal dexamethasone administration induces intrauterine growth retardation and delayed onset of puberty in female rats independent of the hypothalamic Kiss1-gonadotropin-releasing hormone (GnRH) system. The aim of this study was to evaluate the influence of chronic intracerebroventricular (central, CD) or subcutaneous (peripheral, PD) dexamethasone administration to prepubertal female rats on the onset of puberty and body weight change. Rats administered dexamethasone from day 25 to day 34 (CD and PD) showed significantly reduced body weight gain throughout the experimental period and delayed onset of vaginal opening compared with rats administered saline centrally (CS) or peripherally (PS). At 34 days old, hypothalamic Kiss1r mRNA levels were significantly lower with CD than with CS. No significant differences were seen between rats administered saline and rats administered dexamethasone with regard to hypothalamic Kiss1, GnRH and NPY mRNA levels or serum LH levels. Serum leptin concentrations were higher in CD and PD than in the controls (CS and PS). These results suggest that the delayed onset of puberty induced by prepubertal dexamethasone administration occurs independent of the hypothalamic Kiss1-GnRH system.

Delay in the onset of puberty of intrauterine growth retarded female rats cannot be rescued with hypernutrition after birth.

Perinatal undernutrition is known to disturb reproductive development, in particular by delaying the onset of puberty in certain species. Using a rat model, we studied whether hypernutrition after birth can rescue the delayed onset of puberty in intrauterine undernourished female rats. Pregnant rats were divided into two groups: the maternal normal nutrition (mNN, n = 8) and maternal undernutrition (mUN, n = 9) groups. In the mUN group, dams received 50% of the daily food intake of the mNN group from day 15 of pregnancy until delivery. Pups from both the mNN and mUN dams were then separated into two groups, based on their postnatal feeding conditions: control-normal nutrition (control-NN), control-hypernutrition (control-HN), Intrauterine growth retardation (IUGR)-normal nutrition (IUGR-NN), and IUGR-hypernutrition (IUGR-HN). Litter sizes of the hypernutrition groups were controlled to five pups per dam, and normal nutrition groups to 12-13 pups per dam. From postnatal day 30, pups were inspected daily for vaginal opening (VO). The age of VO in the IUGR-NN group was 35.7 ± 2.4 days (mean ± SD), which was significantly delayed compared to that of the control-NN group (33.8 ± 0.8 days). The age of VO in the IUGR-HN group was 35.5 ± 2.3 days, which was significantly delayed compared to that of the control-HN group (33.5 ± 0.8 days). Interestingly, the age of VO did not differ between the IUGR-NN and IUGR-HN groups. In conclusion, maternal undernutrition delays puberty in female offspring, and this delay in puberty cannot be rescued with hypernutrition after birth.

Effects of lipopolysaccharide exposure at different postnatal time points on the response of LH to homotypic stress in adulthood.

Early-life immune stress may have long-lasting effects, known as programming effects, on the physiological response to stress in adulthood. There may be a critical window after birth during which such a challenge can induce long-lasting alterations. However, there are few reports regarding the consequences of this phenomenon for later reproductive function. Here we report on induction by early-life LPS injection of long-lasting alterations in the adult LH response to homotypic immune stress in male rats. First, we investigated developmental changes in the LH response to LPS, since immune challenge during the stress hyporesponsive period can induce long-lasting effects on physiological functions. Rat serum LH concentrations were decreased by LPS (100 µg/kg) injection on postnatal day 15 or 25, but not day 10, suggesting that the period prior to postnatal day 10 is the stress hyporesponsive period for LH. Serum LH concentrations and body weight were decreased by adult LPS (400 µg/kg) injection in rats given saline or LPS (100 µg/kg) on postnatal day 25, but not in rats given LPS (100 µg/kg) on postnatal day 10. Expression of hypothalamic IL-1ß and TNF-α mRNA, which suppress serum LH during immune stress, were equally increased in these groups by adult LPS (400 µg/kg) injection. The present data suggest that the period prior to postnatal day 10 is the critical window in which immune stress can induce long-lasting alterations in the LH response, but that IL-1ß and TNF-α are not involved in mediating the altered response.

Developmental changes in the mammalian gonadotropin-inhibitory hormone (GnIH) ortholog RFamide-related peptide (RFRP) and its cognate receptor GPR147 in the rat hypothalamus.

The mammalian gonadotropin-inhibitory hormone (GnIH) ortholog RFamide-related peptide (RFRP) is considered to act on gonadotropin-releasing hormone (GnRH) neurons and on the pituitary to inhibit gonadotropin release and synthesis. To understand the functional significance of this neuropeptide, we investigated the physiological changes in RFRP at mRNA and peptide levels, as well as at the mRNA level of its cognate receptor, G protein-coupled receptor 147 (GPR147), in the rat hypothalamus during development. We also investigated the effects of gonadal steroids on mRNA expression levels of these molecules. In male rats, mRNA expressions of both RFRP and GPR147 increased from postnatal days 12 and 16, peaking at postnatal days 35 and 42, respectively. However, their expressions fell at postnatal day 49. In female rats, mRNA expression of RFRP continued to increase throughout development; mRNA expression of GPR147 in female rats increased from postnatal day 16, peaking at postnatal day 28, but decreased from postnatal day 35. The hypothalamic contents of RFRP on postnatal days 28 and 42 were significantly higher than on postnatal day 4 in male rats, and those on postnatal day 42 were significantly higher than those on postnatal days 4 and 28 in females. Neither orchidectomy nor ovariectomy influenced mRNA expression levels of RFRP or GPR147 in the prepubertal period when endogenous sex steroid levels were low in males and females. Administration of estradiol-17ß (E2) increased mRNA expression of RFRP in prepubertal females. These results suggest that the hypothalamic RFRP system changes during development. An ovarian sex steroid, E2, may stimulate mRNA expression of RFRP in the prepubertal period when the basal E2 concentration is low.

Fasting reduces the kiss1 mRNA levels in the caudal hypothalamus of gonadally intact adult female rats.

Kisspeptin, which is the product of the kiss1 gene and its receptor kiss1r, have emerged as the essential gatekeepers of reproduction. The present study used gonadally intact female rats to evaluate fasting-induced suppression of the KiSS-1 system of anteroventral periventricular nucleus (AVPV) and arcuate nucleus (ARC) under normal physiological conditions. Starting on the day of estrous, one group of rats was subjected to 72 h of food deprivation, while the other group of rats was able to continue feeding ad libitum. The length of the estrous cycle was significantly longer in the food-deprived rats as compared to the feeding rats. At the end of the 72-h food deprivation period, all of the food-deprived rats were at the diestrous phase, with their serum concentrations of LH and leptin significantly lower than that observed in the feeding rats. In addition, as compared to the feeding rats, the expression levels of kiss1 mRNA were significantly lower in the food-deprived rats in the posterior hypothalamic block, which contained the ARC, but not in the anterior hypothalamic block, which contain the AVPV. However, both the kiss1r mRNA expression levels in the anterior and posterior hypothalamic blocks and the neurokinin B and neurokinin 3 receptor mRNA expression levels in the posterior hypothalamic block were not significantly different between the feeding and food-deprived rats. Thus, lower kiss1 mRNA levels in the ARC appear to be responsible for the fasting-induced inhibition of gonadotrophin secretion and subsequent prolongation of the estrous cycle.

Effect of immune stress on body weight regulation is altered by ovariectomy in female rats.

It has been suggested that obesity and loss of ovarian function alter the inflammatory response to immune stress. Ovariectomized (OVX) rats, which are used as a model of human menopause, exhibit both hyperphagia-induced obesity and gonadal steroid deficiency. To evaluate the effects of ovariectomy on inflammatory responses, we compared the anorectic response to LPS in OVX rats and gonad intact female rats. As leptin and hypothalamic interleukin-1ß (IL1ß) play pivotal roles in the anorectic response to immune stress, these factors were also measured. It was found that the OVX rats exhibited an increased anorectic response to LPS compared with the sham-operated rats. The OVX rats showed higher serum leptin concentrations and a greater increase in hypothalamic IL1ß mRNA expression after LPS injection. In addition, in order to determine whether gonadal steroid deficiency contributes to the changes in the inflammatory responses of OVX rats, we compared responses between OVX rats treated with gonadal steroids and untreated OVX rats. There were no differences in appetite, the serum leptin level, and hypothalamic IL1ß mRNA expression between the two groups after LPS injection. These findings suggest that the loss of ovarian function increases the induction of leptin and hypothalamic IL1ß synthesis and consequently increases the anorectic response under immune stress conditions. It is possible that these alterations are caused by OVX-induced obesity rather than the direct effects of gonadal steroid deficiency.

Changes in responsiveness of appetite, leptin and hypothalamic IL-1ß and TNF-α to lipopolysaccharide in developing rats.

In addition to its role as a regulator of energy homeostasis, leptin plays a pivotal role in some immune/inflammatory responses. Synthesis and secretion of leptin are increased under immune stress conditions, and increased leptin may participate in the development of anorexia and fever. These actions are partially mediated by up-regulation of hypothalamic IL-1ß. Leptin also protects against immune stress-induced lethality. On the other hand, the response and roles of leptin to immune stress conditions in the neonatal period have scarcely been examined. We hypothesized that 1. the response of leptin to immune stress would be suppressed in the early neonatal period, 2. hyporesponse of leptin in the early neonatal period would attenuate the anorectic response and increase the lethal rate under immune stress conditions and 3. supplementation of leptin in the early neonatal period would increase the anorectic response, whereas it would decrease the lethal rate under immune stress conditions. To test these hypotheses, we first examined the developmental changes in the responses of leptin and hypothalamic proinflammatory cytokines, i.e., IL-1ß and TNF-α, to LPS-induced immune stress in female rats. We also examined the developmental changes in the anorectic response and lethality rate under LPS-induced immune stress conditions. Five- and 15-day-old rats showed no leptin response and a weak hypothalamic IL-1ß response to LPS when compared with 25- and 42-day-old rats. Fifteen-day-old rats showed low anorectic responses and high lethality rates when compared with 25- and 42-day-old rats under LPS-induced immune stress conditions. We then examined whether administration of leptin affected the response to the anorectic and lethal effects of LPS in 15-day-old rats. Administration of leptin further attenuated body weight after LPS injection, but not after saline injection. On the other hand, administration of leptin did not affect survival rate. In addition, hypothalamic IL-1ß mRNA levels were not affected by leptin administration. In conclusion, the absence of a leptin response may act to prevent immune stress-induced anorexia during the early neonatal period.

Changes in the responsiveness of serum leptin and hypothalamic neuropeptide Y mRNA levels to food deprivation in developing rats.

Neuropeptide Y (NPY) is an important orexigenic peptide that acts in the brain. The increase in hypothalamic NPY mRNA expression induced by fasting is mainly caused by a decrease in the effects of leptin. We investigated the developmental changes in the sensitivities of leptin and hypothalamic neuropeptide Y to fasting. Hypothalamic NPY mRNA levels were increased by fasting in postnatal days 15 and 25 rats, but not in postnatal day 5 rats. Serum leptin levels were decreased by fasting in rats at all ages (days 5, 15, and 25). In addition, hypothalamic OB-Rb mRNA levels were decreased by fasting in postnatal day 25 rats, but not in postnatal day 5 or 15 rats. Although the percentage of fating-induced decrease in the serum leptin level was larger in the postnatal day 15 rats than in the postnatal day 25 rats, the percentage of increase in the hypothalamic NPY mRNA level in the postnatal day 15 rats was smaller than that in the postnatal day 25 rats. There was a strong inverse correlation between serum leptin levels and hypothalamic NPY mRNA levels in the postnatal day 25 rats, whereas no significant correlation was found between these parameters in the postnatal day 5 or 15 rats. These findings indicate that the sensitivity of hypothalamic NPY mRNA expression to food deprivation and hypoleptinemia has developed by postnatal day 25.

Delayed puberty in prenatally glucocorticoid administered female rats occurs independently of the hypothalamic Kiss1-Kiss1r-GnRH system.

Intrauterine growth retardation (IUGR) is an important risk factor for the pathogenesis of diseases after birth. Long-lasting alterations in the structure and function of tissues and the neuroendocrine system, which are known as 'programming effects', increase the risks of these diseases. To investigate the pathophysiology of programming effects, several kinds of IUGR rodent models have been used in experiments. Sexual maturation and the onset of puberty are delayed in these models. We have previously reported that decreased action of hypothalamic kisspeptin, which is a positive regulator of GnRH, contributes to the delayed onset of puberty in undernutrition-induced IUGR rats. The aim of this study was to evaluate whether the hypothalamic Kiss1-Kiss1r-GnRH system is also altered in dexamethasone-induced IUGR rats. Compared with offspring from an untreated mother (control), offspring from a dexamethasone administered mother (DEX) showed a significant reduction in body weight throughout the experimental period (from birth to the prepubertal period) and the delayed onset of puberty. There were no significant differences between the control and DEX groups with regard to their hypothalamic Kiss1, Kiss1r, GnRH, CRH, NPY and POMC mRNA levels during the experimental period or their serum LH, FSH, or leptin concentrations at postnatal day 28 or vaginal opening (VO). Compared with the control, DEX showed significantly lower ovarian weight at postnatal day 28, but not at VO. These results suggested that the delayed onset of puberty induced by maternal dexamethasone administration would occur independently of hypothalamic Kiss1-Kiss1r-GnRH system.

Sensitivities of mRNA expression levels of Kiss1 and its receptor, Kiss1r, to nutritional status are changed during the developmental period in female rats.

Decreased activity of kisspeptin, the product of the hypothalamic Kiss1 gene, is the major cause of the suppression of reproductive function in subnutritional conditions. The sensitivities of the endocrine and the hypothalamic neuronal systems to nutritional status develop during the neonatal period. We examined the developmental changes in the sensitivity of hypothalamic mRNA expression of Kiss1 and its receptor, Kiss1r, to nutritional status in female rats. Kiss1 mRNA expression was reduced by 24 h food deprivation (24 h FD) at postnatal day 25, but not at postnatal day 5 or 15. Kiss1r mRNA expression was reduced by the 12 or 24 h FD at postnatal days 5 and 25, but not at postnatal day 15. Kiss1r mRNA level was found to be correlated with the plasma leptin level, and the administration of leptin, which increased the serum leptin concentration above the physiological range, restored the acute FD-induced suppression of Kiss1r mRNA expression. These data suggest that the hypothalamic Kiss1 and Kiss1r mRNA expression is differentially affected by the nutritional condition at different age points. It is speculated that the sensitivity of Kiss1 mRNA, which is expressed in kisspeptin neuron, to nutritional status develops during the neonatal period. On the other hand, it seems that the sensitivity of Kiss1r mRNA, which is expressed in GnRH neuron, to nutritional status has been already established during the early neonatal period. These data also show that hypoleptinemia plays a role in the reduction of hypothalamic Kiss1r mRNA expression under subnutritional conditions.