Recent investigations of the physiological roles of CRH-R2 are reviewed and summarized in Fig. 5. VMH CRH-R2 is more important than CRH-R1 in mediating anorexic effect of CRH or urocortin (UCN) and stress-induced reduction of food intake. CRH-R2 mediates a central anxiolytic response, opposing the anxiogenic effect of CRH mediated by CRH-R1. Hippocampal CRH-R1 mediates stress- induced enhancement of learning, while CRH-R2 in the lateral intermediate septum may act to impair learning. CRH-R1 mediates CRH-induced blood pressure elevation, while peripheral CRH-R2 mediates the hypotensive effect of systemically administered UCN and CRH. It is likely that CRH-R2 does not play an important role in hypothalamic-pituitary adrenal axis regulation, though it has been reported that CRH-R2-deficient mice showed hyper-response of ACTH and corticosterone. Peripheral CRH-R2 mediates UCN-induced mast cell degranulation, vascular permeability, and abdominal surgery -induced gastric stasis. These recent investigations have revealed that the existence of two CRH receptors, which mediate some opposite effects, provides the CRH and UCN systems a high flexibility and dynamic role in the adaptation of the body to environmental challenge.

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We have reported that alfacalcidol plus menatetrenone, a vitamin K₂ with four isoprene units (menaquinone -4), treatment is useful for improving bone problems in children with skeletal unloading. The aim of this study was to evaluate the effect of menatetrenone on bone metabolism in long-term glucocorticoid-treated children with alfacalcidol treatment. Twenty children who had been treated with fixed dosages of prednisolone and alfacalcidol (0.03μg/kg/day) for 24 weeks were enrolled in a prospective pilot study, and assigned to receive alfacalcidol (0.03μg/kg/day) or alfacalcidol (0.03μg/kg/day) plus menatetrenone (approximately 2mg/kg/day). Bone biochemical markers and bone mineral density (BMD) were measured at baseline and after the 12-week treatment. In the group receiving alfacalcidol plus menatetrenone, serum carboxylated osteocalcin (OC) (p=0.0022) and lumbar BMD (p=0.0029) increased and serum undercarboxylated OC (p=0.0004) decreased significantly in comparison to the group receiving alfacalcidol; further, the change of lumbar BMD showed an inverse correlation to the change of serum undercarboxylated OC (r=-0.744, p=0.0134) and positive correlations to the baseline values of bone turnover markers such as serum levels of intact OC, bone-specific alkaline phosphatase and type I procollagen carboxyl extension peptide and urinary levels of deoxypyridinoline and N-telopeptide of type I collagen. No adverse effect was observed. This is a small short-term study, but its results suggest that menatetrenone effectively and safely increases lumbar BMD probably through carboxylation of OC in long-term prednisolone-treated children with alfacalcidol treatment who have a high bone turnover. Randomized double-blind controlled trials are needed to confirm our findings.

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In a 37-year-old man who had Cushing's syndrome, investigations, including overnight dexamethasone suppression test, corticotropin-releasing hormone (CRH) test, pituitary MRI and inferior petrosal sinus sampling suggested the presence of ectopic adrenocorticotropin (ACTH) production. Interestingly, gonadotropin-releasing hormone (GnRH) increased plasma ACTH from 73pg/ml to 708pg/ml at 15min. Furthermore, desmopressin also increased plasma ACTH whereas CRH and thyrotropin-releasing hormone (TRH) had no effect. Such paradoxical responses of plasma ACTH were observed repeatedly. A thoracic CT scan revealed a right anterior mediastinal mass, which was surgically resected. Histological and immunohistochemical examination confirmed that the tumor was an ACTH-producing carcinoid. ACTH and cortisol decreased immediately following surgery. Neither desmopressin nor GnRH administration resulted in elevation of plasma ACTH while ACTH-responsiveness to dexamethasone and CRH was restored. To our knowledge, this is the first report documenting GnRH responsiveness in ectopic ACTH syndrome.

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The renin-angiotensin system (RAS) has been unequivocally implicated as a mediator of diabetic complications. The present study was designed to evaluate the RAS in non-insulin dependent diabetic patients with diabetic nephropathy. Plasma renin activity, plasma angiotensin II and serum angiotensin-converting enzyme (ACE) activity were measured in 45 non-insulin dependent diabetes mellitus (NIDDM) patients and 15 healthy non-diabetic controls. Diabetics were subdivided into 15 normoalbuminuric NIDDM subjects, 15 NIDDM patients with microalbuminuria and 15 diabetics with macroalbuminuria. Mean plasma renin activity for macroalbuminuric diabetics (0.65±0.10ng/ml/hr) was significantly reduced than the controls (1.28±0.37ng/ml/hr) (P<0.001), the diabetic group with microalbuminuria (1.08±0.48ng/ml/hr) (P<0.05) and normoalbuminuric patients (1.56±0.82ng/ml/hr) (P<0.001). A significant negative correlation was obtained between serum creatinine and plasma renin activity (r=-0.842, p<0.001) in macroalbuminuric NIDDM patients. Plasma angiotensin II was significantly decreased in non-complicated diabetics compared to healthy controls (4.36±1.49pg/mlvs 14.87±3.48pg/ml respectively, p<0.001). Non-insulin dependent diabetic patients with nephropathy had significantly higher plasma angiotensin II levels (28.99±5.88pg/ml) than non-complicated diabetics (p<0.001). Serum ACE activity was increased in 53.3% of NIDDM patients. All diabetic groups showed increased serum ACE activity (normoalbuminuric NIDDM 114.9±28.3nmol/min/ml, microalbuminuric NIDDM 127.9±31.2nmol/min/ml and macroalbuminuric NIDDM 127.0±29.3nmol/min/ml) when compared to the normal control group (76.3±16.5 nmol/min/ml) (p<0.001). No significant difference in serum ACE activity was obtained between normoalbuminuric and nephropathic diabetics or between diabetics with and without retinopathy. No significant correlation was obtained between serum ACE activity and blood pressure, blood glucose level and duration of diabetes. Thus plasma renin activity is decreased in diabetic nephropathy and negatively correlates with serum creatinine. Plasma angiotensin II is decreased in normoalbuminuric diabetics and elevated in diabetic nephropathy. Serum ACE activity is raised in NIDDM patients with no relation to albumin excretion rate. The role of increased ACE activity in NIDDM remains to be established.

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Earlier studies in platelet aggregation have shown that females seemed to have greater aggregability than males as detected by conventional aggregometry which used light transmission (LT), but controversy still remains. This study was performed to determine whether sex difference exists in platelet aggregation by using the recently developed laser light scattering (LS) method, which can detect small aggregates (i.e., two or three platelets). Blood was drawn from healthy volunteers (10 male and 10 female in follicular phase after menstruation), and platelet aggregation was detected by either LT or LS method in platelet rich plasma. Platelet aggregation was stimulated by increasing concentration of adenosine 5' diphosphate (ADP, 0, 0.5, 1 and 2μM). To detect the effect of sex hormones, platelets were incubated with estradiol (10nM) or testosterone (40nM) for 30min, then platelet aggregation studies were performed. LT method revealed that female had greater aggregability than male. With weak stimuli (_??_1μM ADP), LS method showed that females had more medium aggregates than males, and that testosterone decreased small aggregates, and that estradiol decreased all sizes of aggregates. These data suggest that the female is more conductive to platelet aggregation than the male at a physiologic concentration of ADP (_??_1μM), but that both estradiol (10nM) and testosterone (40nM) have countereffects on platelet aggregation at the same condition. Therefore, the reason why females have greater aggregability than males may partly be explained by their lack of testosterone, but the mechanism still remains to be elucidated.

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A 15-year-old girl with Cushing's disease exhibited adrenal insufficiency following incomplete transsphenoidal resection of a large pituitary corticotropinoma, approximately 35mm in diameter. Within two weeks following surgery, her plasma ACTH level decreased from 42 to 13pmol/l, while, her plasma cortisol levels and urinary excretion of free cortisol decreased from 607nmol/l and 1112nmol/day to 94nmol/l and 55nmol/day, respectively. Immunoreactive ACTH was characterized in plasma using Sephadex G-75 column chromatography and measuring ACTH with immunoradiometric assay (IRMA) and radioimmunoassay (RIA) to determine additional peaks, other than the one demonstrated for 1-39 ACTH. In particular, when measured with RIA, a broad peak including the high molecular weight ACTH was detected as well as 1-39 ACTH. The bioactivity of the high molecular weight ACTH in patient plasma was lower than the reference range of 1-39 ACTH, which is determined by the ability of dispersed rat adrenocortical cells to secrete corticosterone. The large pituitary corticotropinoma found in this patient secreted not only 1-39 ACTH but also high molecular weight proopiomelanocortin (POMC)-derived peptides, which could be detected by measuring with IRMA and RIA for ACTH. Based on the results of biological activity and molecular ratios, no positive evidence could be found to support the hypothesis that the high molecular weight ACTH induced any postoperative adrenal insufficiency in this patient. However, based on this study, the possibility of adrenal insufficiency should be carefully monitored, even when post-operative remnant tumor tissue is clearly present in patients with Cushing's disease, accompanied by macrocorticotropinoma.

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Transsphenoidal surgery is the treatment of choice for ACTH-producing pituitary adenoma (Cushing's disease) and pituitary irradiation is widely considered the most appropriate treatment for patients with Cushing's disease for whom transsphenoidal surgery has been unsuccessful. We studied 49 consecutive patients who underwent transsphenoidal surgery for the treatment of Cushing's disease at Tokyo Women's Medical University from 1977- 1997 with a mean follow-up duration of 87.6 months (range, 24-253 months). We examined the relationship between postoperative endocrinological data, assessed between 3 and 8 weeks after surgery, and long-term outcome and efficacy of pituitary irradiation after surgery. Long-term remission was defined as the regression of the symptom and signs of Cushing's syndrome, and restoration of normal levels of plasma ACTH, cortisol and urinary free Cortisol, together with adequate suppression of morning plasma cortisol levels following the administration of low dose (1mg) of dexamethasone. Thirty patients had no additional treatment after pituitary surgery. Only 1 of 25 patients (4%) whose postoperative plasma cortisol level was less than 2μg/dl developed recurrent disease whereas 3 out of 5 patients with postoperative plasma cortisol levels higher than 2μg/dl relapsed. Postoperative external pituitary radiation was used to treat the remaining 19 patients. Four patients who received radiation therapy had a low or undetectable postoperative plasma cortisol level (<2μg/dl, 56nmol/L) and all of these patients developed hypopituitarism whereas 5 patients with subnormal plasma cortisol levels (2.0-10.0μg/dl) remained in remission. Among 10 patients with persistent disease after surgery, 6 entered remission 6-47 months after irradiation but one of them subsequently relapsed after 108 months. These results suggest that 1) additional therapy should be avoided in patients with a postoperative plasma cortisol less than 2μg/dl because relapse is very rare and radiotherapy will frequently induce hypopituitarism, 2) patients with a subnormal cortisol level following surgery should be treated with pituitary irradiation, because the relapse rate is reportedly high and radiotherapy is effective in preventing relapse, 3) radiotherapy in patients with persistent disease after surgery is effective only in 50% (5/10) of the patients.

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We reported previously that vitamin D deficiency is a causal mechanism of postoperative tetany in patients with Graves' disease. The aim of the present study was to determine the prevalence of vitamin D deficiency by reviewing serum 25(OH)D levels in 208 patients with Graves' disease (146 women, 62 men) during a 1 year period. Serum 25(OH)D levels were significantly lower (p<0.001) in female Graves' patients (31.8±13.3nmol/l) than in male patients (41.3±15.0nmol/l). Vitamin D deficiency (defined as a serum 25(OH)D value below 25nmol/l) was found in 40% of female patients and in 18% of male patients (p<0.005). There was a significant seasonal variation in the 25(OH)D concentrations in female patients [amplitude 6.38 (95% CI, 5.42-7.56)], with values below 25nmol/l found in 58% of female patients during the winter months. There were significant (p<0.001) differences in serum 25(OH)D levels between age groups in the female patients. The concentrations were lowest in patients in their twenties (25.1±8.2nmol/l) and highest in patients in their fifties and sixties (43.2±13.7nmol/l). Serum 25(OH)D concentrations might be monitored in patients with Graves' disease during antithyroid drug therapy, and vitamin D and/or calcium supplements are recommended for patients with vitamin D deficiency.

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In the present study we searched for prolactin receptor (PRL-R) in porcine ovarian theca tissue (Tc) of small, medium and large follicles, as well as in early corpus luteum (ECL). The objectives of this investigation were: 1) comparison of the direct effect of PRL action on progesterone (P₄) and estradiol (E₂) secretion from Tc and ECL cells in culture with adequate effects caused by luteinizing hormone (LH). 2) detection of the presence and distribution of PRL-R in thecal tissue of porcine follicles and in ECL. Tissues were cultured as monolayers either in control M199 medium with calf serum or in medium either with PRL (100ng/ml) or with LH (100ng/ml). After 2 days in vitro cultured media were assayed for steroid concentrations by radioimmunoassays. Content and distribution of PRL-R were evaluated by Scatchard analysis and by an immunohistochemical assay. Separated theca layers as well as fragments of ECL were excised on dry ice, homogenized, and incubated with [¹²⁵I]-PRL. PRL stimulated P₄ secretion from Tc 10-fold versus controls. LH stimulated P₄ secretion only 2.5-fold. E₂ secretion was stimulated by PRL 2.7-fold and by LH 2.4-fold. LH enhanced P₄ secretion from ECL cells by 18% while PRL increased P₄ secretion by as much as 73%. Femtomol amounts of PRL-R protein were detected in theca tissues of medium and large follicles and also in ECL, which was in accordance with immunohistochemical results. The results showed for the first time the presence of PRL-R in porcine Tc and ECL.

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To examine the clinical significance of the insulin resistance index as determined by homeostasis model assessment (HOMA-IR), we investigated the relationship between HOMA-IR and the insulin resistance estimated by the euglycemic-hyperinsulinemic clamp method in various subgroups and compared the significance of HOMA-IR with that of fasting plasma insulin levels (FIRI). HOMA-IR was significantly correlated to the inverse of the glucose infusion rate (1/GIR) in both diabetic and non-diabetic subjects (r=0.747, P<0.0001 and r=0.419, P<0.002, respectively). In the diabetic patients, treatment with sulfonylureas did not weaken this correlation (r=0.833, P<0.0001). HOMA-IR was found to be closely related to FIRI (r=0.932, P<0.0001), but HOMA-IR was more closely associated with 1/GIR than FIRI was. HOMA-IR as well as 1/GIR was correlated with the visceral fat area (VFA) more closely than with the subcutaneous fat area (SFA), while FIRI was correlated almost equally with both of them. In conclusion, HOMA-IR is a convenient and beneficial method for evaluating insulin resistance, especially in subjects with visceral fat accumulation, and reflects insulin resistance obtained by euglycemic clamp more accurately than FIRI alone.

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Plasma leptin concentration is closely associated with body fat in humans, with energy restriction inducing a greater decrease in plasma leptin than in body fat. Since adequate energy restriction is mandatory in diet therapy of diabetes mellitus especially in obese subjects, the present study was undertaken to evaluate the clinical implication of serial leptin measurement in the management of diabetic patients. Fifty-four consecutive subjects with type 2 diabetes, who were subjected to adjusted energy restriction during hospitalization, were enrolled in the study. During their hospitalization period (24±4 days), plasma leptin concentrations decreased from 6.9±0.7 to 5.7±0.6pg/l (P<0.0001) in the overall subjects, and the %change in plasma leptin (-13.9%) was greater than the %changes in body mass index (BMI) and percent body fat (-1.7% and -4.7%, respectively). The %change in plasma leptin was positively correlated with the %changes in BMI and plasma C-peptide (r=0.526, P<0.0001 and r=0.446, P<0.002, respectively) and negatively with a %change in plasma ketone bodies (r=-0.516, P<0.005). Multiple regression analysis revealed that the %changes in BMI and plasma C-peptide were independent determinants of the %change in plasma Leptin. In addition, 38 subjects were followed up after discharge. Three months after discharge, plasma leptin concentrations significantly increased by 25.6%, which was again much greater than the %change in BMI (+0.9%). In 28 subjects who showed increase in plasma leptin levels after discharge, BMI was also increased. In contrast, the remaining 10 subjects without the increase in plasma leptin kept their BMI unchanged. Throughout the observation period, the changes in plasma leptin were prominent in the subjects with BMI greater than 25kg/m². In conclusion, plasma leptin concentrations showed greater changes than the alterations in anthropometric indexes during the observation period. Serial leptin measurement may be useful to estimate adherence to energy restriction especially in obese subjects with type 2 diabetes.

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In this study, we elucidated the cellular localization of somatostatin receptor (SSTR) by immunohistochemistry using an antibody specific for SSTR type 2A (SSTR2A) in various organs of rat and human. SSTR2A expression was basically similar in rat and human, except in the pancreas and adrenal cortex. In the pituitary gland, the posterior lobe and the majority of growth hormone cells and some ACTH and TSH cells expressed SSTR2A. In rat adrenal gland, the zona glomerulosa strongly expressed SSTR2A, whereas zone-specific immunoreactivity was not observed in human. The adrenal medulla moderately expressed SSTR2A in both rat and human. SSTR2A immunoreactivity was observed in islet cells and some ductal cells in human pancreas, and also in acinar cells of rat The number of SSTR2A positive cells was much more than that of chromogranin A positive endocrine cells. In the kidney, the glomerular capillaries and collecting bules, but not proximal tubules, showed immunoreactivity. SSTR2A immunoreactivity was observed not only in endocrine cells but also in non-endocrine cells.

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A rare case of aldosterone-producing adrenocortical adenoma with preclinical Cushing's syndrome and hypersecretion of parathyroid hormone (PTH) is described. A 64-year-old male patient had a history of hypertension for two decades and hypokalemia for 4 years. He suffered from left hemiparesis and aphasia due to cerebral hemorrhage, but his appearance was not Cushingoid. His plasma reamn activity was below the normal range, while plasma aldosterone concentration was high. They did not respond to furosemide-upright test. His plasma cortisol level in the morning was at the upper limit of the normal range, but it did not show a diurnal rhythm nor was it suppressed by 1mg and 8mg of dexamethasone. Computed tomography showed a low density tumor in the right adrenal gland. An adrenal scintigram under dexamethasone treatment revealed an uptake of the tracer on the right side, and plasma aldosterone and cortisol concentrations in the adrenal vein were higher on the right side than on the opposite. The diagnosis of right aldosterone-producing adrenal adenoma with an autonomous production of cortisol was confirmed by right adrenalectomy. Histological findings showed an adenoma consisting mostly of clear cells, but that the nests of compact cells were scattered. Analysis of an extract from the adenoma revealed that the adenoma contained an excess amount of aldosterone and that the cortisol/corticosterone ratio was higher than that of aldosterone-producing adenoma. Both serum calcium and PTH levels remained high one year after adrenalectomy. Ultrasonography revealed the swelling of a parathyroid gland on the left side, indicating the coexistence of an utonomous hyperparathyroidism.

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Although heterozygous lipoprotein lipase (LPL) deficiency is not rare, only part of the phenotypes may have been reported in Japan. Here we describe a Japanese family with LPL_Arita, the most common mutation linked to familial LPL deficiency in Japan, and show for the first time a heterozygote for the mutation who had marked hypercholesterolemia due to increased low-density lipoprotein (LDL) cholesterol. The proband's mother, one of the eterozygotes for LPL_Arita in the family, had both severe hypercholesterolemia (total cholesterol 306mg/dl) with an especially increase in LDL-cholesterol and mild hypertriglyceridemia (180mg/dl). She had normal LDL receptor activity and did not show clear evidence of possible causes of secondary hyperlipidemia. In addition to being heterozygous for LPL deficiency, she was also heterozygous for apo ε₄. Because the ε₄ allele is known to be associated with higher LDL-cholesterol, heterozygous apo ε₄ may be one of causes of her LDL-cholesterol elevation. The other three heterozygotes for LPL_Arita were moderate drinkers, and all of them had both remarkable hypertriglyceridemia and mild hypercholesterolemia due to increased very-low-density lipoproteins (VLDL). The results suggest that heterozygotes for LPL_Arita can exhibit various phenotypes of hyperlipidemia, that is, hypertrigliceridemia and/or hypercholesterolemia due to not only increased VLDL but also increased LDL. The phenotypes appear to depend on some other genetic and environmental factors.

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The regulation of renal 11β-hydroxysteroid dehydrogenase type 2 (11βHSD2) gene expression is poorly understood. Inhibition of expression can result in hypertension. An example of this is in ectopic adrenocorticotropin (ACTH) syndrome (EAS). Inhibition of 11βHSD2 activity is suggested by the observed increased ratio of cortisol to cortisone in both plasma and urine. To investigate whether ACTH or ACTH-dependent steroids can modulate renal 11βIHSD2 gene expression we analysed renal 11βHSD2 mRNA levels after treatment with ACTH of 1H and 24H and demonstrated no change in the levels of gene expression. We have demonstrated in this study that the expression of 11βIHSD2 in the kidney is unaltered by ACTH. The reduced inactivation of cortisol by 11βIHSD2 observed in EAS is likely to be in part due to end product inhibition or substrate overload of the enzyme by endogenous substrates (cortisol, corticosterone, etc) rather than inhibition of 11βHSD2 at the transcriptional level by either ACTH or ACTH regulated steroids.

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