G protein-coupled receptor 30/G protein-coupled estrogen receptor-1 (GPR30/GPER-1) was reported as a novel membrane receptor for estrogen in 2005. However, the research on GPR30 has produced conflicting reports with regard to its intracellular localization, the tissue distribution of its expression, and some its functions. Recently, in addition to the finding of G-1, a GPR30 agonist, GPR30 KO mice have been produced in laboratories, and this has significantly increased the confidence in the data. In this review, the intrinsic appearance of GPR30 is approached based mainly on data obtained in vivo.

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Cytoplasmic calcium ([Ca²⁺]_i) provided through voltage-dependent Ca²⁺ channels (VDCC) plays an important role in adrenocorticotropin (ACTH)-induced steroidogenesis in adrenocortical cells. To identify alternative mechanisms for [Ca²⁺]_i supply, we investigated the 2-aminoethoxydiphenyl borate (2APB)-sensitive pathway as one of the possible signaling pathways involved in [Ca²⁺]_i supply for ACTH-induced steroidogenesis. In monolayers of cultured rat adrenal fasciculate and reticularis cells, ACTH at 10-11 M stimulated corticosterone synthesis without increasing intracellular cAMP, and corticosterone synthesis was decreased by 10 μ M 2APB by 51.8% (6.71 ± 0.97 vs. 3.23 ± 0.05 ng/mL/4hours; p<0.05). Furthermore, 2APB significantly decreased the 10^-11 M ACTH-stimulated [Ca²⁺]_i. ACTH increased the intracellular inositol-1,4,5-trisphosphate (IP3) content with a peak at 10^-13 M ACTH, which illustrates the possibility that ACTH activates IP3/diacylglycerol- dependent protein kinase C signal transduction. However, the difference in ACTH concentrations between that responsible for the IP3 increase and steroidogenesis without elevated cAMP, suggest a hypothesis that IP3 is not required for steroidogenesis, but does involve an unknown messenger, which stimulates the release of Ca²⁺ from the ER or the subsequent store-operated Ca²⁺ entry (SOCE). The pregnenolone concentration in the culture medium was increased by ACTH, which was significantly suppressed by 2APB, showing that the 2APB-sensitive Ca²⁺ supply affects cholesterol transport into the mitochondrial membrane via steroidogenic acute regulatory protein. Therefore, the SOCE may contribute to ACTH-induced steroidogenesis in the mitochondrial region. In conclusion, the [Ca²⁺]_i used for steroidogenesis may be derived from a 2APBsensitive pathway and via VDCCs, particularly at physiological concentrations of ACTH. We suggest that ACTH receptors activate steroidogenesis via inositol triphosphate, or an unknown downstream messenger, which could be inhibited by 2APB.

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Impaired secretion of glucagon-like peptide 1 (GLP-1) has been suggested to contribute to the deficient incretin effect in patients with type 2 diabetes mellitus (T2DM). Recent studies, however, have not always supported this notion. Since Japanese patients with T2DM usually have severe impairment in the earlyphase of insulin secretion, the measurement of incretin secretions in Japanese T2DM patients would be useful for assessing the association between incretin levels and insulin secretion. We conducted an oral glucose tolerance test (75 g) (OGTT) and meal tolerance test (480 kcal) (MTT) for subjects with normal glucose tolerance (NGT, n=12), subjects with impaired glucose tolerance (IGT, n=7), and T2DM patients (n=21). The tests were carried out over 120-min study periods on separate occasions. Intact GLP-1, GIP, and dipeptidyl peptidase (DPP)-IV were measured by ELISA. T2DM exhibited an impaired early phase of insulin secretion and a reduction in glucagon suppression. There were no significant differences in GLP-1 or GIP levels at each sampling time among NGT, IGT, and T2DM after the ingestions; hence the incremental areas under the curve (IAUC) for the three groups were quite similar. The levels of DPP-IV, a limiting enzyme for the degradation of incretins, were comparable among the three groups. The GLP-1-IAUC was not correlated with IAUCs of insulin, C-peptide, or glucagon determined by the OGTT or the MTT. We concluded that intact GLP-1 levels are comparable between non-diabetics and T2DM, suggesting that impaired insulin secretion in Japanese T2DM is not attributable to defect in GLP-1 secretion.

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The present study examined the gonadotropin regulation of pituitary adenylate cyclase-activating polypeptide (PACAP) and PACAP type I receptor (PAC₁-R) expression, and its role in progesterone production in the human luteinized granulosa cells. The stimulation of both PACAP and PAC₁-R mRNA levels by LH was detected using a competitive reverse transcription-polymerase chain reaction (RT-PCR). PACAP transcript was stimulated by LH reaching maximum levels at 12 hours in a dose dependent manner. LH treatment also stimulated PAC₁-R mRNA levels within 24 hours. Addition of PACAP-38 (10^-7 M) as well as LH significantly stimulated progesterone production during 48 hours culture. Furthermore, co-treatment with PACAP antagonist partially inhibited LH-stimulated progesterone production. Treatment with vasoactive intestinal peptide, however, did not affect progesterone production. Taken together, the present study demonstrates that LH causes a transient stimulation of PACAP and PAC₁-R expression and that PACAP stimulates progesterone production in the human luteinized granulosa cells, suggesting a possible role of PACAP as a local ovarian regulator in luteinization.

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In order to evaluate the relationship between clinical markers of glycemia and glucose excursion, we performed 48-hour continuous glucose monitoring (CGM) in 43 diabetic patients. For the clinical markers, HbA_1c, glycoalbumin (GA), and 1,5-anhydroglucitol (1,5-AG) were measured, and for the parameters of glucose excursion from CGM, average glucose (AG), standard deviation of glucose (SD), the area under the curve for glucose levels >180 mg/dL (AUC_>180), and the difference between the maximum and minimum glucose levels during 48 hours (ΔG_48hr) were analyzed. All patients were treated without any changes of the dosages of oral anti-diabetic agents or insulin for at least the previous 3 months with coefficient of variation (CV) of HbA_1c less than 4 %. In results, while HbA_1c did not show any single correlation with AG, SD, AUC_>180, or ΔG_48hr, both GA and 1,5-AG were significantly related to all these parameters. Furthermore, GA significantly correlated to all CGM parameters, and SD significantly correlated to GA in multiple regression analyses. These results suggest that GA may be a different marker from HbA_1c for diabetic complications, because GA, but not HbA_1c, may reflect not only short-term average glucose but also fluctuation of glucose.

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Adenosine 5’ -monophosphate-activated protein kinase (AMPK) has been implicated in the regulation of energy metabolism, although its role in the pancreatic β cells remains unclear. In the present, we have overexpressed a dominant negative form of AMPKα1 subunit (Asp57Ala) tagged with c-myc epitope (AMPKα1-DN) in INS-1D cells with an adenoviral vector. After 48 h of adenoviral infection, overexpression of AMPKα1-DN in INS-1D cells was confirmed by Western blot analysis with anti-c-myc antibody. Phosphorylation of the Thr172 in AMPKα1/α2 subunit was progressively decreased in parallel with increasing number of adenoviral titers. Glucose-stimulated insulin secretion in response to 30 mmol/L glucose was decreased in INS-1D cells overexpressing AMPKα1-DN as compared to control cells infected with adeno- LacZ vector. Neither cellular insulin content nor insulin mRNA level was changed between the two groups. Phosphorylation of acetyl-CoA carboxylase (ACC), a down-stream substrate of AMPK, was decreased, indicating that ACC activity was increased, due to the decreased AMPK activity. In fact, intracellular triglyceride content was increased as compared to control cells. The β-oxidation of palmitate was decreased at 30 mmol/L glucose. Insulin secretion in response to potassium chloride or glibenclamide was also decreased as compared to control cells. In conclusion, suppression of AMPK activity in β-cells inhibited insulin secretion in response to glucose, potassium chloride or glibenclamide without altering insulin content. Accumulation of triglyceride subsequent to the activation of ACC by suppression of AMPK activity, was suggested to be, at least in part, responsible for the impaired insulin secretion through so called lipotixicity mechanism.

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To investigate genes modulated in the parathyroid glands by calcium, expression levels of mRNA for all genes expressed in parathyroid tissue explants (PTEs) obtained from patients with primary hyperparathyroidism (I°-HPT) were analyzed by oligo-DNA microarray. PTEs obtained from 4 patients with I°-HPT were precultured in normocalcemic medium (Ca⁺⁺ 1.0-1.1 mM) for 7 days and then cultured in hypocalcemic medium (Ca⁺⁺ 0.60 mM) or hypercalcemic (Ca⁺⁺ 1.60 mM) medium containing 4mg/dl phosphate for an additional 7 days. As expected, expression levels of mRNA for PTH and chromogranin A were decreased to less than 50% in the hypercalcemic medium when compared with those in the hypocalcemic medium. Furthermore, oligo-DNA microarray analyses revealed that 7 genes were up-regulated by more than 2-fold and more than 30 genes were down-regulated by more than 1/2 in PTEs. Interestingly, 9 of these genes (up-regulated genes: chemokine ligand 8, multiple C2 domain and transmembrane region protein 1; down-regulated genes: matrix metallopeptidase-9, B-box and SPRY domain-containing protein, nitric oxide synthase 2A, PTH, cartilage acidic protein 1, chromogranin A, and fibrin 1) were involved in calcium metabolism or calcium-signaling pathways in the parathyroid tissue. However, the expression level of mRNA for α-klotho was variable, and it was not constantly decreased in hypercalcemic medium under the present experimental conditions. Although it was not possible to use normal parathyroid tissue, this is the first reported study to have investigated the expression levels of mRNA for all genes in human parathyroid adenomas that are modulated by high calcium concentration in organ culture.

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Elevation of serum parathyroid hormone (PTH) in patients with medullary thyroid cancer (MTC) is usually found in multiple endocrine neoplasia type 2A (MEN2A). However, ectopic production of PTH is rare and its molecular etiology remains largely uninvestigated. We report a case of ectopic production of PTH by a sporadic MTC. The etiology of ectopic PTH gene expression was examined, focusing on GCM2 which has a crucial role in developing parathyroid glands. We observed ectopic expression of the PTH and GCM2 genes in tissues from the tumor and metastatic lymph nodes. However, GCM2 gene expression was also detected in adjacent thyroid tissue and lymphoblasts, in which PTH gene expression was absent. Hypomethylation of the PTH promoter, which is reportedly associated with ectopic production of PTH, was not seen in either the tumor tissue or metastatic lymph nodes. Meanwhile, DNA hypomethylation was seen in a CpG island identified in the GCM2 promoter region, regardless of whether or not the GCM2 gene was expressed. We showed that transcriptional activity of the CpG island sequences cloned into a reporter plasmid was dependent upon DNA methylation. Finally, we present the first report of a PTH-producing MTC. There was no apparent association between ectopic PTH and GCM2 gene expression, despite co-expression of the two genes. Neither genomic rearrangement nor DNA hypomethylation in the PTH gene appeared responsible for ectopic production of PTH. Although DNA hypomethylation may be necessary for the GCM2 gene expression, ectopic expression of GCM2 won’t be possible by DNA hypomethylation alone.

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We report on a Japanese girl with HDR (hypoparathyroidism, sensorineural deafness, and renal dysplasia) syndrome who developed diabetes mellitus (DM) at three years of age (blood glucose 713 mg/dL, HbA_1c 8.0%) in the absence of anti-glutamic acid decarboxylase autoantibodies. Mutation analysis revealed a de novo heterozygous two base pair deletion at exon 6 of the GATA3 gene (c.1200_1201delCA; p.H400fsX506). GATA3 expression was identified by PCR amplification for human pancreas cDNA, and mouse Gata3 was weekly but unequivocally expressed in pancreatic β cells. The results, in conjunction with the previous findings indicating the critical role of GATA3 in lymphocyte function, GATA3 haploinsufficiency may affect the function of β cells and/or lymphocytes, leading to the development of DM in relatively exceptional patients with high susceptibility to DM.

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Ghrelin has a stimulating effect on arginine vasopressin (AVP). However, it is not known whether GHRP-2, a synthetic ghrelin receptor agonist, also has a stimulating effect on AVP release in men. To determine whether the GHRP-2 test is useful for assessing AVP secretion, blood ACTH, GH, FSH, LH, PRL, TSH and AVP levels, as well as glucose, osmolality, sodium and hematocrit, were measured before and 15, 30, 45 and 60 min after an intravenous bolus of 100 μg GHRP-2 in 10 healthy men with and without fasting. Blood pressure was measured at 15-min intervals. AVP secretion was not stimulated by the GHRP-2 test with and without fasting. There were no significant differences in hematocrit, blood pressure and plasma osmolality before and after GFRP-2 injection, although significant (p<0.001) peak blood GH, and ACTH and PRL levels were observed 30 and 15 min after GHRP-2 injection with and without fasting, respectively, and the maximal peaks were significantly (p<0.05) higher with fasting than without fasting. These results suggest that AVP secretion is not stimulated by the GHRP-2 test both with and without fasting, though GH, ACTH and PRL levels were higher with than without fasting.

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Type 1 diabetic patients who have endured their condition for prolonged periods are not uncommon, but there are few well-documented cases of type 2 diabetic patients with duration of over fifty years. In the present case study, we analyzed the history of a diabetic patient whose duration was 53 years. Her case was consequently diagnosed not as the common type 2 diabetes, but as the slowly progressive type 1 diabetes (SPIDDM) identified by Japanese medical researchers. The patient, now 73 years old, was first diagnosed with diabetes in 1953 when she was 17 years of age and started insulin injections. In 1962 she was referred to our hospital, and two years later she vaginally delivered a healthy baby (birth weight 3100 g) at the 40^th week of gestation. She was the first case of a diabetic mother delivering an infant treated at Tokyo Women’s Medical College Hospital. Her data shows that her C-peptide responses by meal tolerance test in 1978 was at least partly preserved though it decreased year by year. Her anti-GAD antibody was found to be positive in 2000 and remained so in 2009. This leads us to conclude that the etiology of her SPIDDM was most likely has insulin secretion exhaustion.

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