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Orexins are neuropeptides that express primarily in the hypothalamus and are produced in two isoforms, orexin A and orexin B. There are two kinds of G protein coupled receptors, orexin type 1 (OX1R) and type 2 (OX2R) receptors. Orexin has been reported to have key roles on sleep-wake regulation and feeding behavior in the central nervous system, whereas its receptors are also expressed in peripheral tissues including the endocrine organs, and orexin affects the regulation of the endocrine system. In our previous experiments, we have revealed the impact of orexins on anterior pituitary functions. For instance, we reported that orexin A plays an inhibitory role in prolactin production through the suppression of endogenous bone morphogenetic protein (BMP) activity in rat pituitary lactotrope GH3 cells. It was also reported that orexin A enhances pro-opiomelanocortin (POMC) transcription by upregulating corticotropin-releasing hormone (CRH) receptor signaling and by downregulating BMP-Smad signaling in mouse corticotope AtT20 cells. However, the effects of orexin on the endocrine function regarding gonadotrope cells remain unclear. We have recently uncovered that core clock genes and BMPs have mutual effects on luteinizing hormone (LH) expression in a phase-dependent manner by mouse gonadotrope LβT2 cells. In the present study, we investigated the effect of orexin on pituitary gonadotropin expression using LβT2 cells, which express OX1R and OX2R, by focusing on the functional involvement of BMP system and clock genes. It was revealed that orexin A stimulation increased LHβ and FSHβ mRNA expression in a concentration-responsive manner in the absence of GnRH, and interestingly, GnRH co-treatment further upregulated LHβ mRNA expression in LβT2 cells. Regarding the interrelationships between the signalings of orexin and BMPs, it was also revealed that orexin A pretreatment enhanced the BMP receptor signaling detected as the Smad1/5/9 phosphorylation, indicating that orexin enables to upregulate the BMP actions in LβT2 cells. In our previous studies, we reported that several BMP ligands such as BMP-6, -7 and 15 expressed in LβT2 cells can promote gonadotropin transcription, in which BMP-6 regulates GnRH-stimulated LH expression by modulating the sensitivity to somatostatin analogs. Based on the present results, it was implied that endogenous orexin can be functionally involved in the underlying mechanisms of gonadotropin expression. Collectively, orexin enhances gonadotropin expression by regulating BMP signaling in gonadotrope cells. Here, we will also discuss functional involvement of clock genes in the regulatory system of gonadotropin secretion induced by orexin and BMPs. Presentation: No date and time listed

Primary hyperparathyroidism (PHPT) is the most common disorder causing hypercalcemia, in which parathyroid hormone (PTH) is excessively secreted from a parathyroid tumor. PHPT occurs mainly in elderly women, and patient with PHPT present symptoms including nephrolithiasis, bone fracture, and cognitive impairment or can be asymptomatic. Excessive secretion of PTH leads to elevated serum levels of alkaline phosphatase as well as calcium and decreased serum inorganic phosphate level. Parathyroid surgery is the only established treatment, and the treatment reduces elevated serum levels of calcium and PTH, increases bone mineral density, reduces the occurrence of bone fractures, and decreases the risk of renal stones. However, some PHPT patients, especially elderly patients, refuse to undergo surgical therapy for various reasons including perceived risk. Calcimimetic treatment, including cinacalcet and evocalcet administration, has also been reported to be effective for reducing serum calcium level in patients with PHPT. However, the relationship between the efficacy of calcimimetic treatment for PHPT and patient characteristics related to the effectiveness of calcimimetics remains to be elucidated. To investigate the relevance of clinical parameters to the effectiveness of calcimimetics for PHPT, we retrospectively analyzed patients with PHPT who received calcimimetic treatment in our department. We included nine patients including 8 females (88.9%) and one male (11.1%) in the study. The median age of the patients was 81 years (interquartile range (IQR): 61-86 years). Laboratory tests before calcimimetic treatment revealed that median level of serum corrected calcium (cCa) was high (11.1 (IQR: 10.5-12.7) mg/dL), serum inorganic phosphate was low (2.3 (2.3-2.4) mg/dL), and plasma intact PTH was high (251. 0 (198.3-498.5) pg/mL). The fractional excretion of calcium was higher than 1% (1.3% (1.12-1.48%)), %tubular reabsorption of phosphate was low (79.9% (77.1-83.9%)), nephrogenous cyclic adenosine monophosphate (cAMP) was high (3.4 (2.4-4. 0) nmoL/dL GF), and urinary cAMP was normal (4.3 (4. 0-5.3) μmoL/day). Median reduction rate of serum cCa level due to calcimimetic treatment was 5.8% (0-13.4%) at the first follow-up (median treatment duration: 22 (20-29) days). Notably, reduction rate of serum cCa level had significant correlations with age (R=0.95), dual energy X-ray absorptiometry % young adult mean (DEXA %YAM) in the femoral neck (R=-0.92), and urinary cAMP level (R=-0.85). Thus, the present study suggested that elevated serum cCa levels may be reduced by treatment with calcimimetics more effectively in PHPT patients of relatively advanced age, patients with low DEXA %YAM, and patients with low urinary cAMP. Presentation: No date and time listed

The pandemic of the new coronavirus infection (COVID-19) has yet not only to be converged, but also new infections continue to occur. At least about one-third of the affected patients suffer from various symptoms even after the acute phase. However, effective treatments for sequelae have not yet been established, and the prognosis and risk factors are still unclear. It is possible that endocrine disruption is involved in the occurrence of long COVID. Okayama University Hospital set up a COVID-19 aftercare (CAC) outpatient clinic on Feb. 15 in 2021 to treat patients suffering from sequelae even one month after COVID-19 infection. In the present study, to extract the key factors that affect the onset and course of sequelae, a retrospective analysis was performed by focusing on changes in the number of symptoms and patient background during the three months from the first outpatient visit. Also, we focused on how various sequelae progress in patients who received continuous medical care for 3 months, in which the relationship between the sequelae and the serum COVID-19 antibody titer measured at the first visit was evaluated. In this study, the data from 65 patients who visited our clinic. Presentation: No date and time listed

Leptin and ghrelin are the two major hormones which regulate food intake and energy balance. They have also been reported to have pleiotropic functions, including the modulation of ovarian function via the hypothalamic-pituitary-ovarian system or in a direct manner to the ovaries. Blood leptin levels increase over the menstrual cycle and peak at the time of the luteinizing hormone (LH) surge. Leptin secreted from adipocytes acts on the hypothalamus and pituitary gland to stimulate the secretion of GnRH and gonadotropins. In the ovaries, high levels of leptin inhibit follicle-stimulating hormone (FSH)-induced steroid secretion and follicular development. Ghrelin, secreted by the stomach, has been reported to modulate GnRH secretion in the hypothalamus and to affect FSH and LH secretion, as well as steroidogenesis in the ovaries. Knockout of these hormones leads to impaired fertility. However, the mechanism of the direct effects of leptin and ghrelin on the ovaries has not been well elucidated. In the present study, we investigated the effects of leptin and ghrelin on the ovarian steroidogenesis using primary culture of rat granulosa cells, by focusing on the ovarian bone morphogenetic protein (BMP) system, which plays an important role in follicular development and steroidogenesis and acts as a luteinizing inhibitor. The results showed that treatment with leptin (1 to 300 ng/mL) did not affect either estradiol (E2) or progesterone (P4) production, but enhanced FSH-induced P4 production at low concentrations (10 ng/mL). On the other hand, treatment with ghrelin (1 to 300 nM) did not affect E2 and P4 production, but enhanced FSH-induced P4 production in a concentration-responsive manner. The mRNA levels of the follicular steroid synthases (StAR, P450scc, 3βHSD, 20αHSD) were examined. Leptin treatment enhanced 3βHSD mRNA levels. Both leptin treatment and ghrelin treatment decreased FSH-induced 20αHSD mRNA levels. However, both treatments decreased FSH-induced cAMP production. In addition, leptin treatment tended to suppress the transcriptional activity of Id-1 induced by BMP-6. Collectively, these results indicate that both leptin and ghrelin enhance FSH-induced P4 production by suppressing BMP signaling in rat granulosa cells. It was thus suggested that leptin and ghrelin which regulate food intake and energy balance can also act as endogenous regulators for the modulation of P4 synthesis and luteinizing process by ovarian follicles. Presentation: No date and time listed

Vitamin D is obtained in the body by food intake or by production from 7-dehydrocholesterol by exposure of the skin to ultraviolet B radiation. It is first metabolized in the liver to 25-hydroxyvitamin D (25D), which is a major circulating metabolite. In the kidney, 25D is subsequently metabolized to the hormonally active form, 1,25-dihydroxyvitamin D (1,25D), via 1α-hydroxylase encoded by the CYP27B1 gene. 1,25D has a cellular effect through the vitamin D receptor, which leads to calcium absorption in the gut, bone metabolism, and parathyroid function. A recent study showed that a low vitamin D status is common worldwide and is associated with various diseases including kidney, heart, and liver failure, secondary hyperparathyroidism, osteomalacia, inflammatory bowel disease, granuloma-forming disorders (sarcoidosis and tuberculosis), and cancer. Vitamin D deficiency also increases the risks of falls, fractures, bone loss, sarcopenia, leading to worse outcomes of illness severity, morbidity, and mortality. The 1,25D/25D ratio is considered to be a useful tool for diagnosis of ocular sarcoidosis; however, its clinical utility and relevance to pathophysiology of evaluation of the ratio 1,25D/25D which indicates vitamin D activation have remained unknown. To clarify the clinical usefulness of markers for vitamin D activation, 87 patients in whom serum 25D and 1,25D level was measured were retrospectively reviewed in the present study. Data for 79 patients (33 males and 46 females) were analyzed after exclusion of 8 patients taking vitamin D. The median serum 1,25D/25D ratio was significantly lower in males than in females: 4.1 (IQR: 2.3–5.8) x 10−3 versus 6.8 (3.0–9.8) x 10−3. However, individual levels of 25D and 1,25D were not different in males and females. The major categories of main disorders were endocrine (30.6 %), inflammatory (18.5 %), and bone-related (16.7 %) disorders. The ratios of serum 1,25D/25D had significant negative correlations with femoral dual energy X-ray absorptiometry % young adult mean (DEXA %YAM) (R=-0.35) and lumbar DEXA %YAM (R=-0.32). Significant correlations were found between 1,25D/25D ratio and serum levels of inorganic phosphate (R=-0.34), intact parathyroid hormone (R=0.64) and alkaline phosphatase (R=0.46) in all patients. Of interest, the 1,25D/25D ratio had gender-specific characteristics: the ratio had a significant correlation with age in males (R=0.49), while it had a significant correlation with body mass index (BMI) in females (R=0.34). Collectively, the results revealed that the ratio of serum 1,25D/25D as a marker for activation of vitamin D had relevance to clinical parameters, especially bone turnover, with gender-specific features. It is suggested that the existence of a gender-specific difference of aging males and obese females regarding the activation of vitamin D that is functionally linked to bone metabolism.

Corticosteroids are important bioactive substances in the body that regulate inflammation, metabolism, immunity, and circulatory dynamics. Cortisol and aldosterone are two major naturally occurring steroids, which synthesized in the adrenal fasciculata and in the adrenal glomerulosa under the regulation of adrenocorticotropin (ACTH) and renin-angiotensin system (RAS), respectively. The conventional strategy for evaluating adrenocortical function has been to measure random serum cortisol or 24-hour stored levels of urine free cortisol (UFC). UFC is a more appropriate marker to avoid the effects of diurnal fluctuation of cortisol, serum cortisol-binding proteins, and cortisol clearance in the kidney. Thus, measurement of UFC has been a reliable test for diagnosis of Cushing’s disease or adrenal insufficiency. However, since the normal range of UFC varies widely, it is often difficult to evaluate whether the UFC level is optimal or not for each patient. That is because UFC is greatly affected by the amount of fluid intake or urine volume, and an immunoassay for UFC, which is the usual method for measuring UFC, is susceptible to interference from other steroid metabolites and synthetic glucocorticoids. To explore an alternative indicator, we tried to standardize the levels of UFC by the levels of urinary aldosterone concentration (UAC) in the same urinary sample. Medical records of all 246 patients in whom daily excretions of UFC and UAC had been measured between 2015 and 2018 at our department were reviewed. 142 patients (including 93 females) were included after exclusion of 104 patients because of corticosteroid replacement therapy. UFC/UAC ratio showed significant positive and negative correlations with the levels of serum cortisol (R=0.287) and aldosterone (R=-0.762), respectively. UFC/UAC ratio increased with aging in female patients, while the ratio was not altered by the levels of BMI in either gender. Markers for metabolic and inflammatory status including hemoglobin A1c (R=0.327), albumin (R=-0.331), chorine esterase (R=-0.248), C-reactive protein (R=0.317), ferritin (R=0.473), and D-dimer (R=0.569) showed correlations to the ratio of UFC/UAC that were more significant than the correlations to the serum level of cortisol or UFC alone. Of note, the UFC/UAC ratio was shown as an indicator for risks of diabetes (AUC: 0.765), hypoalbuminemia (0.839), hyper-CRPemia (0.748), and thrombophilia (0.824), in which the cut-off levels of UFC/UAC ratios were found to be around 12. These results indicate that the UFC/UAC ratio is a suitable variable for the detection of metabolic and inflammatory complications related to adrenocortical dysfunction.

We previously reported that the expression levels of Clock gene are linked to the expression levels of steroidogenetic enzymes in human granulosa cells (EJ 2019). However, the downstream molecules of the Clock gene actions in the regulation of ovarian steroidogenesis have yet to be elucidated. In the present study, we investigated the roles of the transcription factors, NR5A1 (also known as SF-1) and NR5A2 (LRH-1), which play key roles in the reproductive function as well as steroidogenesis by focusing on the functional link between Clock gene and bone morphogenetic protein (BMP) signaling using human granulosa KGN cells. First of all, we examined the effects of BMPs/growth differentiation factor (GDF) on forskolin (FSK)-induced steroidogenesis. As a result, FSK-induced mRNA levels of StAR and P450scc, but not P450arom, were potently suppressed by treatments with BMP-6, -9, -15 and GDF-9. The expression levels of NR5A1 and NR5A2 mRNA were also upregulated by FSK treatment, while the BMP-target gene Id-1 mRNA levels were stimulated by the treatment with BMPs. Of interest, treatments with BMPs/GDF increased FSK-induced NR5A1 mRNA levels but suppressed FSK-induced NR5A2 mRNA levels by granulosa cells. The expression levels of NR5A1 mRNA were positively correlated with the changes of P450arom and 3βHSD mRNA, whereas the expression levels of NR5A2 mRNA were correlated with that of StAR and P450scc mRNA. In addition, the expression levels of NR5A1 and NR5A2 mRNAs were positively correlated with the levels of Clock mRNA. In particular, Clock mRNA levels showed highly positive correlation with the levels of NR5A2 mRNA compared with NR5A1 mRNA. Of note, Id-1 mRNA levels were positively correlated with the levels of NR5A1 mRNA, but negatively correlated with that of NR5A2 mRNA. Furthermore, the inhibition of Clock gene expression by siRNA attenuated the expression levels of NR5A1 and NR5A2 mRNA, resulting in decreased mRNA levels of StAR and P450arom in the presence of FSK. Thus, the present results suggested a novel mechanism by which Clock expression is functionally linked to the expression of NR5A1 and NR5A2, the latter of which is further regulated by BMP signaling by granulosa cells. The interaction among Clock, NR5A1/NR5A2 and BMPs may be involved in the fine tuning of steroidogenesis by ovarian follicles.

Osteoporosis is known as a disease that can increase the risk of fracture because of low bone mineral density (BMD). Bone fracture is associated with an increase in mortality in the elderly. It has been reported that femoral bone fracture occurred in 11,578 people in Japan every year during the period from 2008 to 2013. However, in the Japanese population, the average rate of osteoporosis examination is less than 5%. Because of the low rate of consultation for bone examination, it is difficult for general practitioners to detect an early stage of osteoporosis. According to the Ministry of Health, Labour and Welfare, in the current super-aged society in Japan, escalating healthcare costs over the past few decades have been a major problem for maintaining the universal health insurance coverage. Approximately 60% of the total national medical care expenditure is for people over 65 years of age. In Japan, there are about 13 million osteoporosis patients. This causes an increase in medical expenses for elderly and bone fractures lead to an increase in the risk of mortality. In order to reduce the incidence of bone fractures, efforts should be made to reduce the number of patients with low BMD. If physicians can pay attention to the influencing factors in losing BMD, they should be able to detect osteoporosis in an early stage. Earlier detection of low BMD and its treatment would result in reduction in the incidence of bone fractures in elderly patients. In the present study, we attempted to clarify the risk factors that are involved in lowered BMD for patients with various clinical backgrounds who visit Department of General Medicine of a University Hospital, Japan. We retrospectively reviewed medical records for 149 patients who visited our department in our hospital and underwent a BMD examination by the dual energy X-ray absorbance method during the period from 2014 to 2015. We analyzed various physical and laboratory factors that might be associated with lowered BMD. There were 39 patients diagnosed with osteoporosis based on the T-score. Adjusted analysis showed that female gender, aging and increased levels of serum calcium were statistically related to lowered femoral BMD, whereas body mass index was associated with an increase in femoral BMD. In conclusion, it is important for general practitioners to examine BMD in patients who may have low BMD in the viewpoints of gender and aging. Attention should also be given to the possibility of low BMD, patients with high serum calcium levels, patients with anorexia and malnutrition, and patients taking antipsychotic/sleeping drugs or proton pump inhibitors.

Many local factors expressed in the ovary are involved in folliculogenesis. Among these, the ovarian renin-angiotensin system is critical for normal processes of follicle development, steroidogenesis and ovulation. In various clinical conditions such as secondary hypertension, heart failure, and liver and/or renal damages, hyperaldosteronism can be detected. In addition, Aldo levels are known to be high in patients with PCOS and/or OHSS. However, the effect of high concentration of Aldo on reproductive function has not been fully elucidated. In this study, we examined the influence of Aldo on follicular steroidogenesis using primary culture of rat granulosa cells, by focusing on the ovarian bone morphogenetic protein (BMP) system acting as a luteinizing inhibitor. Granulosa cells isolated from female rats were treated with follicle stimulating hormone (FSH) in the presence of low to high concentrations of Aldo (1-300 nM) and cultured in serum-free conditions. After incubation with Aldo for 48 hours, changes in estradiol (E2) and progesterone (P4) productivity induced by FSH stimulation were measured by CLIA, and cAMP synthesis was evaluated by EIA. Total RNA was extracted and mRNA level of steroidogenetic enzymes induced by FSH and that of BMP-receptors and Id-1 induced by BMP-6 were quantified by real-time RT-PCR. Phosphorylation of Smad1/5/9 was further evaluated by Western blotting using cell lysate after the co-treatments with Aldo and BMP-6. As a result, treatment with Aldo increased FSH-induced P4 production by granulosa cells in a concentration-dependent manner at the maximum effects by 100-300 nM of Aldo. Aldo treatment increased mRNA levels of P4-synthetic enzymes expression including StAR, P450scc and 3βHSD, and Aldo also upregulated FSH-induced cAMP synthesis. On the contrary, Aldo failed to change E2 production stimulated by FSH. Moreover, Aldo treatment attenuated Id-1 transcription and phosphorylation of Smad1/5/9 induced by BMP-6 stimulation. These data indicated that Aldo elicits a specific effect on the enhancement of P4 production by granulosa cells. On the other hand, BMP-6 treatment downregulated MR expression in granulosa cell, suggesting the existence of a negative feedback loop between BMP-receptor and MR signaling. Taken it into consider that BMP ligands act as specific suppressor for P4 production as a luteinizing inhibitor, it is possible that Aldo is functionally linked to the suppression of endogenous BMP-receptor signaling in granulosa cells, leading to the deterioration of follicular growth and ovulation process. Collectively, it is revealed that high concentration of Aldo specifically enhances FSH-induced P4 production by inhibiting BMP signal in granulosa cells. This effect may be associated with impaired fertility and/or ovulatory failure in the patients with hyperaldosteronism.

In mammals, the circadian pacemaker is known to control and integrate physiological rhythms via the coordination of oscillating peripheral clocks that reside in various organs, through the biologic actions of various neurochemical and/or hormonal transmitters. The expression of clock genes has been demonstrated in tissues composing the axis of the hypothalamic-pituitary-gonadal system. The roles of hypothalamic oscillators to produce the biological timings and rhythms in the reproductive axis have been recognized. Results of recent studies have shown that clock genes are expressed in the ovary, and accumulated findings have demonstrated a significant interrelationship between clock gene expression and follicular development. It has been reported that the rhythms of clock gene expression are undetectable in primordial and preantral follicles but can be detected at the timing of early antral stages of follicles and then become clearer at the late antral stages to preovulatory follicles. In the ovarian follicles, clock genes are expressed in granulosa cells, theca cells, oocytes and stromal cells composing the developing follicles. Dysfunction of clock gene expression in the ovary and/or desynchronicity between ovarian clocks and extra-ovarian circadian oscillators may cause various reproductive abnormalities. However, the regulatory mechanism and the physiological roles of the intrinsic clock activity in the ovary have remained uncertain. In the preset study, functional link between clock gene expression and ovarian steroidogenesis was studied using human granulosa KGN cells. Similarities between the oscillating patterns of mRNA and protein expression of Bmal1 and Clock and that of Per2 and Cry1 were demonstrated during 24-h culture of KGN cells in the presence of forskolin. Among the interrelationships between the expression levels of clock and steroidogenic factors, Clock mRNA had a strongly positive correlation with P450arom and a negative correlation with 3βHSD. Knockdown of Clock gene by siRNA resulted in a significant reduction of estradiol production by inhibiting P450arom expression, while it induced a significant increase of progesterone production by upregulating 3βHSD in KGN cells treated with forskolin. Moreover, BMP-7 had an enhancing effect on the expression of Clock mRNA and protein in KGN cells. Collectively, the expression levels of Clock, being upregulated by forskolin and BMP-7, were functionally linked to estradiol production and progesterone suppression by human granulosa cells.