Takako Kawami

Hypothalamic Kiss1 and RFRP gene expressions are changed by a high dose of lipopolysaccharide in female rats

Reproductive function is suppressed by several types of stress. Hypothalamic kisspeptin, which is a product of the Kiss1 gene, and GnIH/RFRP have pivotal roles in the regulation of GnRH and gonadotropins through their receptors Kiss1r and GPR147 in many species. However, alterations of these factors under stress conditions have not been fully evaluated. This study investigated the mechanisms of immune stress-induced reproductive dysfunction, especially focusing on the changes of Kiss1 and RFRP gene expression. Serum LH levels and hypothalamic Kiss1 and GnRH mRNA levels were decreased, while hypothalamic RFRP and GPR147 mRNA levels were increased by administration of a high dose of LPS (5mg/kg) in both ovariectomized and gonadal intact female rats. In this condition, Kiss1 and/or RFRP mRNA levels were positively and negatively correlated with GnRH expression, respectively. In contrast, hypothalamic Kiss1, RFRP, and GPR147 mRNA levels were not changed by administration of a moderate dose of LPS (500μg/kg) in ovariectomized rats. Rats with high-dose LPS injection showed more prolonged fever responses and severe anorexia compared with rats with moderate-dose LPS injection, indicating that more energy was used for the immune response in the former. These results suggest that the underlying mechanisms of dysfunction of gonadotropin secretion are changed according to the severity of immune stress, and that changes of some reserved factors, such as kisspeptin and RFRP, begin to participate in the suppression of GnRH and gonadotropin in severe conditions. As reproduction needs a large amount of energy, dysfunction of gonadotropin secretion under immune stress may be a biophylatic mechanism by which more energy is saved for the immune response.

Changes in the responsiveness of hypothalamic prokineticin 2 mRNA expression to food deprivation in developing female rats

Prokineticin 2 (PK2) is highly expressed in several regions of the central nervous system, including the hypothalamus. Recently, it has been suggested that PK2 plays a role in appetite regulation. In adult male rodents, the administration of PK2 decreased food intake, and PK2 mRNA expression was reduced by food deprivation. Usually, the changes in the expression levels of appetite‐regulating factors induced in response to fasting are not fully established during the neonatal period. Thus, we investigated the developmental changes in hypothalamic PK2 mRNA expression and the alterations in hypothalamic PK2 mRNA expression induced by fasting during the pre‐pubertal period in female rats. The changes in hypothalamic neuropeptide Y (NPY) mRNA expression were also examined because NPY is a potent appetite regulatory factor. Hypothalamic PK2 mRNA expression was extremely high during the early neonatal period (postnatal day (PND) 5) compared with that observed during subsequent periods (PND15, 25, and 42), while hypothalamic NPY mRNA expression did not differ among any of the examined periods. A fasting‐induced reduction in hypothalamic PK2 mRNA expression was observed on PND5, but no fasting‐induced increase in hypothalamic NPY mRNA expression was seen during the same period. In addition, the fasting‐induced reduction in hypothalamic PK2 mRNA expression observed on PND5 was more marked than that seen on PND25. These results suggest that the sensitivity of hypothalamic PK2 expression to undernutrition develops during the early neonatal period, when the responses of other appetite regulatory factors to such pressures remain immature.

The responses of hypothalamic NPY and OBRb mRNA expression to food deprivation develop during the neonatal-prepubertal period and exhibit gender differences in rats.

Neuropeptide Y (NPY) is an important hypothalamic orexigenic neuropeptide that acts in the brain. It has been established that the fasting‐induced up‐regulation of NPY expression is mainly caused by a reduction in the activity of leptin, which is a hormone secreted by adipose tissue. We have reported that in female rats hypothalamic NPY mRNA expression does not respond to fasting during the early neonatal period, but subsequently becomes sensitive to it later in the neonatal period. In this study, we compared the developmental changes in the responses of NPY and leptin expression to fasting between male and female rats during the neonatal to pre‐pubertal period. Fasting was induced by maternal deprivation during the pre‐weaning period (postnatal days 10 and 20) and by food deprivation during the post‐weaning period (postnatal day 30). Hypothalamic NPY mRNA expression was not affected by fasting on postnatal day 10, whereas it was increased by fasting on postnatal day 20 and 30 in both males and females. On the other hand, the serum leptin level was decreased by fasting at all examined ages in both sexes. Namely, hypothalamic NPY mRNA expression was not correlated with the reduction in the serum leptin level at postnatal day 10 in either sex. Under the fasted conditions, the hypothalamic NPY mRNA levels of the males were higher than those of the females on postnatal days 20 and 30, whereas no such differences were observed under the normal nourishment conditions. The serum leptin levels observed under the fasted conditions did not differ between males and females at any examined age. These results suggest that some hypothalamic NPY functions develop during the neonatal period and that there is no major difference between the sexes with regard to the time when NPY neurons become sensitive to fasting. They also indicate that hypothalamic NPY expression is more sensitive to under‐nutrition in male rats than in female rats, at least during the pre‐pubertal period.

The suppressive effect of immune stress on LH secretion is absent in the early neonatal period in rats.

Some physiological functions display weak responses to stress in the early neonatal period; i.e., they exhibit stress hyporesponse periods. In this study, we evaluated whether gonadotropin regulatory factors exhibit stress hyporesponsive periods in male and female rats. Rats were intraperitoneally injected with lipopolysaccharide (100 μg/kg) (LPS group) or saline (control group) on postnatal day (PND) 5, 10, 15, or 25. Then, their serum luteinizing hormone (LH) concentrations and hypothalamic mRNA levels of gonadotropin regulatory factors; i.e., kisspeptin (Kiss1), the kisspeptin receptor (Kiss1r), and gonadotropin‐releasing hormone (GnRH), were measured at 2 h after the injection. The hypothalamic mRNA levels of pro‐inflammatory cytokines were also measured because they suppress gonadotropin secretion. The serum LH concentration of the LPS group was lower than that of the control group at PND25 in both sexes, but no such difference was seen at PND5, 10, or 15 in either sex. In both sexes, the hypothalamic tumor necrosis factor (TNF)α and interleukin (IL)‐6 mRNA expression levels of the LPS group were higher than those of the control group at PND25, but not at PND5 or 10. The hypothalamic IL‐1β mRNA expression level of the LPS group was higher than that of the control group at all time points. The hypothalamic Kiss1, Kiss1r, and GnRH mRNA expression levels of the LPS and control groups did not differ at any time point in either sex. These findings suggest that gonadotropin regulatory factors exhibit stress hyporesponse periods. The hypothalamic–pituitary–gonadal axis (HPG) might become responsive to immune stress between PND15 and 25, which could be related to enhanced hypothalamic cytokine expression. The avoidance of infectious stress during the early neonatal period might be important for normal development of the HPG axis.

Changes in leptin production/secretion induced in response to septic doses of lipopolysaccharides in gonadally intact and ovariectomized female rats

In addition to its role as a regulator of energy homeostasis, leptin plays a pivotal role in certain immune/inflammatory responses. It has been reported that the synthesis and secretion of leptin are increased during immune stress in male experimental animals, whereas the changes in leptin synthesis and secretion induced by immune stress in females have not been fully elucidated. In this study, we found that during lipopolysaccharide (LPS)-induced immune stress the synthesis and secretion of leptin were decreased in gonadally intact female rats, but increased in ovariectomized rats. However, the LPS-induced increase in the serum leptin level observed in the ovariectomized rats was partially attenuated by estradiol supplementation. These results suggest that the changes in leptin synthesis and secretion induced in response to immune stress in females are affected by the gonadal steroid milieu and that estradiol and other factors are involved in these alterations.

Insulin Resistance and Metformin Treatment in Women with Polycystic Ovary Syndrome

Abstract: Insulin resistance is one of the key factors in the pathogenesis of polycystic ovary syndrome (PCOS). Obesity, visceral fat accumulation and excessive serine phosphorylation of the insulin receptor are factors responsible for insulin resistance in PCOS. Insulin resistance induces compensatory hyperinsulinemia, which stimulates androgen synthesis in the theca cells of the PCOS ovary. Therefore, hyperinsulinemia results in hyperandrogenemia in PCOS patients who tend to have androgen-producing ovaries. In Japanese PCOS, 25% of patients are obese and 30% of patients have insulin resistance. Improvement of insulin resistance by weight loss, exercise and insulin sensitizing drugs such as metformin can recover reproductive function. Meformin has the potential to induce ovulation in PCOS, and 58% of clomiphene resistant Japanese PCOS patients resume ovulation after combined clomiphene metformin treatment. Clomiphene-metformin therapy is simple and has low risks of multiple pregnancy and ovarian hyperstimulation syndrome. Metformin use should be considered fertility treatment, especially as a second line therapy for the clomiphene resistant PCOS.

The effects of ovariectomy and LPS-induced endotoxemia on resistin levels in female rats

Resistin is involved in the inflammatory response, as well as in insulin resistance. In rodents, resistin levels are partially regulated by ovarian hormones. Thus, ovariectomy-induced changes in resistin levels and their response to lipopolysaccharide (LPS)-induced septic stress were evaluated. Ovariectomized (OVX) rats exhibited higher serum resistin concentrations and visceral and subcutaneous white adipose tissue (WAT) resistin mRNA levels than sham-operated (sham) rats under the saline-injected (basal) conditions. The serum resistin levels of the gonadal intact male rats were higher than those of the sham rats, whereas the serum resistin levels of the male and OVX rats did not differ. In both the sham and OVX rats, the serum resistin concentration and the resistin mRNA levels of WAT were increased by LPS injection. At 24h after the LPS injection, no difference was detected in the serum resistin concentrations or WAT mRNA resistin levels between the sham and OVX rats. These results suggest that ovarian hormones partially regulate the basal resistin levels of female rats.