Tatsuya Nishioka

Psychological stress increased corticotropin-releasing hormone mRNA and content in the central nucleus of the amygdala but not in the hypothalamic paraventricular nucleus in the rat

The central administration of corticotropin-releasing hormone (CRH) to experimental animals sets into motion a coordinated series of physiological and behavioral events that promote survival during threatening situation. A large body of evidence suggest that CRH in the central nucleus of the amygdala (CEA) induces fear-related behaviors and is essential to fear conditioning; however, evidence of CRH-mediated activation of the amygdala under physiological situation is still limited. We report here a study of the impact of a psychological stressor on hypothalamic and amygdala CRH systems in the rat. Non-footshocked rats placed in a floored compartment surrounded by footshocked rats were defined as the psychological stress group. Rats were exposed to psychological stress for 15 min, and then sacrificed 1.5 and 3 h after cessation of stress. We found that our psychological stressor induced an increase in both CRH mRNA levels, as assessed by in situ hybridization histochemistry, and CRH content, as assessed by micropunch RIA, in the CEA. Exposure to the psychological stressor also caused a significant increase in CRH mRNA levels with a trend for an increase in CRH content in the dorsolateral subdivision of the bed nucleus of the stria terminalis (BNST) which is anatomically associated with the CEA. In contrast, psychological stress induced a small, but significant increase in type-1 CRH receptor (CRHR-1) mRNA in the hypothalamic paraventricular nucleus (PVN), while it failed to elevate either PVN CRH mRNA levels or content, CRH content in the median eminence (ME), or levels of plasma ACTH or corticosterone (CORT). Thus, in the context of a psychological stressor, the activation of the amygdala CRH system can occur without robust activation of the hypothalamic CRH system. In the light of previous data that the psychological stress-induced loss of sleep was reversed by the central administration of a CRH antagonist, these data suggest that CRH in the CEA may contribute to the psychological stress-evoked fear-related behavior such as hyperarousal. These data also indicate that in response to a psychological stressor, the amygdala CRH system is much more sensitive than is the CRH system emanating from the PVN.

Urocortins and corticotropin releasing factor type 2 receptors in the hypothalamus and the cardiovascular system

In addition to urocortin (Ucn I), Ucn II and Ucn III were identified as endogenous ligands for corticotropin-releasing factor type 2 receptor (CRF2 receptor). CRF2 receptor is abundantly located in central hypothalamic ventromedial nucleus (VMH) and in peripheral cardiovascular system. In this mini-review, we focused on the roles of these urocortins and CRF2 receptor in the hypothalamus and the cardiovascular system. Ucn II mRNA was increased in the parvocellular part or the magnocellular part of the hypothalamic paraventricular nucleus (PVN) following immobilization stress or 3 days of water deprivation, respectively. Therefore, it is thought that Ucn II may modulate CRF and vasopressin synthesis in the PVN in a paracrine or autocrine fashion through PVN CRF2 receptor. The early and later phases of Ucn I-mediated feeding suppression may be CRF1 and CRF2 receptor-mediated events, respectively. Ucn II decreases food intake at a later phase, beyond 4 h post injection. A large dose of corticosterone increased plasma leptin and insulin levels as well as the levels of CRF2 receptor mRNA. Adrenalectomy, starvation, and immobilization each lowered plasma leptin and insulin levels and were associated with decrements in CRF2 receptor mRNA levels in the VMH. Peripheral injection of leptin increased VMH CRF2 receptor mRNA, as can induce reductions of food intake and body weight, indicating that circulating leptin is involved in the regulation of VMH CRF2 receptor mRNA expression. Therefore, it is also plausible that VMH CRF2 receptor transduces the anorexogenic effects of leptin as well as those of urocortins. The systemic administration of Ucn II decreases mean arterial pressure (arterial vascular tone) and causes tachycardia via vascular CRF2 receptor in rats, similar to the effects of Ucn I. Thus, CRF2 receptor seems to mediate cardioprotective effects of urocortins.

Glucocorticoid Effects on the Diurnal Rhythm of Circulating Leptin Levels

It is known that circulating leptin shows diurnal variation with a nocturnal rise; however, the mechanisms generating this rhythm have not been fully elucidated. Glucocorticoids are a potent stimulator of leptin secretion, and there is a reciprocal relationship between circulating leptin and glucocorticoid levels. We hypothesized that glucocorticoids could modulate the diurnal rhythm of circulating leptin. We therefore explored the diurnal variation of leptin under situations in which subjects showed no or some shift of glucocorticoid diurnal rhythm, such as prednisolone-administered humans, and adrenalectomized and corticosterone-replaced (ADX+B) rats. The peak level of plasma cortisol immunoreactivity was shifted from early morning to noon by prednisolone administration. The nocturnal increment of plasma leptin in prednisolone-administered patients (71.2 ± 14.2% from 08:00 h value) was significantly greater than that in normal volunteers (12.2 ± 7.5% from 08:00 h value), but the timing of nadir and the peak of plasma leptin was not shifted. In normal rats, the plasma concentration of leptin showed the diurnal rhythm with the bottom at 16:00 h and the top between midnight and early morning. The amplitude of leptin diurnal rhythm was significantly reduced in ADX+B rats (08:00 h: 3.0 ± 0.2, 16:00 h: 2.7 ± 0.2, 00:00 h; 3.7 ± 0.2 ng/ml) compared with sham operated rats (08:00 h: 3.0 ± 0.2, 16:00 h 2.2 ± 0.2, 00:00 h: 4.7 ± 0.4 ng/ml); but ADX+B rats still retained similar timing of nadir and the peak of plasma leptin as observed in sham rats. These results indicate that glucocorticoids enhance the amplitude of leptin diurnal rhythm, and are consistent with previous findings showing that glucocorticoids increase leptin secretion. Glucocorticoids appear to play modulatory, but not essential roles in generating leptin diurnal rhythm.

Sitagliptin, a Dipeptidyl Peptidase-IV Inhibitor, Improves Psoriasis

A patient with a 17-year history of plaque psoriasis accompanied by type 2 diabetes mellitus discontinued cyclosporine and steroid ointment given for treatment of psoriasis because she was dissatisfied with the effects of the drugs. After sitagliptin, a dipeptidyl peptidase-IV (DPP-IV) inhibitor, was administered for control of blood glucose, psoriatic skin lesions were gradually diminished, although HbA1c did not improve. Three months after the administration of sitagliptin, infiltration, scales and erythema on all psoriatic plaques disappeared, leaving pigmentation on flat skin. DPP-IV in serum degrades the incretin hormones which stimulate β-cell insulin secretion. DPP-IV inhibitors, as incretin enhancers, cause an increase in glucose-dependent insulin secretion, and are applied for the treatment of diabetes mellitus. DPP-IV is also expressed on T cells as CD26, a surface antigen which plays an important role in activating T cells. As helper T cells are involved in the pathogenesis of psoriasis, it is possible that DPP-IV inhibitors improve psoriatic skin lesions by inhibiting T cell activation, independently of glycemic control. DPP-IV inhibitors could be an alternative for the treatment of psoriasis.

Regulation of interleukin-1 receptors and hypothalamic-pituitary-adrenal axis by lipopolysaccharide treatment in the mouse.

We measured iodine-125-labeled recombinant human interleukin-1 alpha (125I-IL-1 alpha) binding in the hippocampus, pituitary, liver, spleen and testis, and plasma adrenocorticotropic hormone (ACTH) and corticosterone levels after i.p. injection of various dose and treatment regimens of the bacterial endotoxin, lipopolysaccharide (LPS). Plasma ACTH and corticosterone levels were significantly increased at 2 h after acute administration of LPS (60 or 300 micrograms/mouse). 125I-IL-1 alpha binding in all peripheral tissues examined was significantly and comparably decreased at 2 h after a single injection of 30 micrograms or 300 micrograms LPS/mouse. On the other hand, 125I-IL-1 alpha binding in hippocampus was significantly decreased only after high dose administration of LPS (300 micrograms/mouse). In order to evaluate if activation of IL-1 in brain resulting in the observed decrease in 125I-IL-1 alpha binding may require more sustained exposure to endotoxin, we compared the effects of a single injection (60 micrograms/mouse) and two injections of LPS (30 micrograms/mouse each at 0 and 12 h). A single injection of LPS (60 micrograms/mouse) decreased 125I-IL-1 alpha binding in the testis but not in the hippocampus, while two LPS injections (30 micrograms/mouse each at 0 and 12 h) caused dramatic reductions in 125I-IL-1 alpha binding in both the hippocampus and testis.(ABSTRACT TRUNCATED AT 250 WORDS)

Central type 2 corticotropin-releasing hormone receptor mediates hypothalamic-pituitary-adrenocortical axis activation in the rat.

In an attempt to clarify the role of the type 2 corticotropin-releasing hormone (CRH) receptor (CRHR-2) in the brain in activation of the hypothalamic-pituitary-adrenocortical axis, we conducted experiments using male Wistar rats. First, an injection of urocortin-2 (7.5 µg) into the lateral ventricle resulted in transient increases in CRH heteronuclear RNA (hnRNA) in parvocellular paraventricular nucleus (PVN) and in plasma adrenocorticotropic hormone (ACTH), whereas sustained increases in arginine vasopressin (AVP) hnRNA and c-fos mRNA in the parvocellular PVN were observed as compared with vehicle treatment. Pretreatment with the selective CRHR-2 antagonist antisauvagine-30 (20 µg) into the lateral ventricle 15 min prior to agonist injection attenuated the stimulatory effects of urocortin-2 on the above-mentioned hypothalamic-pituitary-adrenal axis variables. These effects were similar or rather more potent than those induced by pretreatment with 50 µg of α-helical CRH. Second, we found longer-lasting increases in CRH and AVP hnRNA and c-fos mRNA in parvocellular PVN and in plasma ACTH following central administration of urocortin-3 (7.5 µg) than following urocortin-2. Pretreatment with antisauvagine-30 antagonized the effects of urocortin-3 on the above-mentioned variables. Finally, central administration of antisauvagine-30 as well as α-helical CRH profoundly attenuated restraint-stress-induced increases in AVP hnRNA. However, α-helical CRH, but not antisauvagine-30, attenuated restraint-stress-induced increases in CRH hnRNA in the PVN. Both antagonists transiently attenuated stress responses of c-fos mRNA in PVN and plasma ACTH. These results indicate that there is a CRHR-2-mediated mechanism in the brain that stimulates CRH- and AVP-producing neurons in the PVN which results in the promotion of plasma ACTH secretion.

Significant Gene Expression of Insulin-Like Growth Factor II and Proliferating Cell Nuclear Antigen in a Rapidly Growing Recurrent Pituitary ACTH-Secreting Adenoma

Background: We quantified the expression of various growth-related factors in an adrenocorticotropic hormone (ACTH)-secreting adenoma that had recurred very rapidly as invasive macroadenoma. Methods/Results: A 43-year-old woman underwent successful transsphenoidal surgery for Cushing’s disease. Seven years later, she was admitted to our ward for further endocrine examinations. In spite of a very high plasma ACTH level, the serum cortisol level was normal. Discrepancies between ACTH and cortisol levels were detected on the basis of diurnal rhythms, dexamethasone suppression tests, and corticotropin-releasing hormone test. The patient showed no clinical features of Cushing’s disease. Magnetic resonance imaging of the pituitary showed an almost empty sella, and no microadenoma was found. These results, along with those of Sephadex column gel filtration and high-performance liquid chromatography of plasma-immunoreactive ACTH, suggested that the patient’s residual corticotrophs secreted biologically inactive ACTH. Two years later, the patient suddenly developed diplopia and right abducens nerve palsy. She was slightly moonfaced and centrally obese. Her plasma ACTH and serum and urinary free cortisol levels were elevated, although discrepancies between ACTH and cortisol still existed. Magnetic resonance imaging revealed a large pituitary mass with suprasellar and cavernous sinus extensions. The tumor was excised, and the proopiomelanocortin gene and the expression of growth-related factors were analyzed. No mutations were found in the ACTH-coding region of the proopiomelanocortin gene. A significant expression of insulin-like growth factor II and proliferating cell nuclear antigen mRNAs was demonstrated. A high MIB-1 antibody labeling index was also detected in the adenoma tissue, suggesting high Ki-67 expression. Conclusion: These growth- and proliferation-related factors might be involved in the rapid growth and aggressiveness of this patient’s pituitary adenoma.

Corticotropin-releasing factor treatment upregulates interleukin-1 receptors in the mouse pituitary : reversal by dexamethasone

Intraperitoneal injection of rat/human corticotropin-releasing factor (CRF) (40 micrograms/kg/0.2 ml of saline) resulted in a dramatic increase in specific iodine-125-labeled human interleukin-1 alpha ([125I]IL-1 alpha) binding in the male C57BL/6 mouse pituitary at 2 and 6 h after the injection although it did not affect [125I]IL-1 alpha binding in the mouse hippocampus, spleen and testis at any time after the injection. [125I]IL-1 alpha binding was unchanged at 2 h following dexamethasone (DEX) treatment (1 mg/kg/0.2 ml of 4% ethanol-saline) in the mouse pituitary and the hippocampus. In contrast, DEX inhibited CRF-induced upregulation of IL-1 receptors in the pituitary at 2 h after the injection. These data demonstrate complex interactions between CRF and DEX on IL-1 receptors during stress.

Biallelic inactivation by somatic mutations of the MEN1 gene in sporadic parathyroid tumors

We report three sporadic parathyroid tumors with biallelic inactivation of the multiple endocrine neoplasia type 1 (MEN1) gene. Three parathyroid tumors had two somatic mutations (K119del and 864del8, 363insT and 1767delT, and 508del33 and W341X, respectively). The mutations in both alleles detected by long-range polymerase chain reaction and subcloning in three tumors would likely result in a nonfunctional menin protein in parathyroid glands. These results show that the MEN1 gene is inactivated not only by a combination of somatic mutations and loss of heterozygosity, but also by somatic double mutations located on different alleles. The results directly confirmed the participation of MEN1 in the tumorigenesis of sporadic parathyroid tumors.

Effects of adrenomedullin on adrenocorticotropic hormone (ACTH) release in pituitary cell cultures and on ACTH and oxytocin responses to shaker stress in conscious rat.

Adrenomedullin (AM) is a potent vasodilator peptide, which is initially isolated from tissue of human pheochromocytoma. In addition to the effect on cardiovascular system, previous studies suggest that AM plays some roles as a neuropeptide in the brain. In the present study, we examined the effect of AM on in vitro adrenocorticotropic hormone (ACTH) secretion stimulated by corticotropin-releasing hormone (CRH), vasopressin (VP) or oxytocin (OT) in cultured rat corticotrophs and on the response of plasma ACTH, corticosterone (B) and OT to shaker stress in vivo. In contrast to the previous report, basal or CRH (10(-9) M)-stimulated ACTH secretion was not affected by coincubation with AM. Either of VP (10(-8) M) or OT (10(-8) M) significantly increased ACTH secretion in cultured rat anterior pituitary cells (156.7+/-24.9 in basal incubation vs. 267.8+/-15.0 in VP-stimulation, P<0.05, and 308.6+/-41.3 pg/ml in OT-stimulation, P<0.05). AM (10(-10) M) significantly inhibited OT-stimulated ACTH secretion. AM tended to inhibit VP-stimulated ACTH secretion, although the inhibitory effect was not statistically significant. Thus, it is likely that AM attenuates OT-stimulated ACTH secretion in corticotrophs. In vivo study, male Wistar rats were prepared with a guide cannula in the lateral ventricle and a catheter in femoral artery for blood sampling. AM (0.5, 1.0 microg in 5 microl) or normal saline (5 microl, control) was intracerebroventricularly (i.c.v.) injected in conscious rats. Shaker stress (110 cycles/min for 5 min) produced a significant increase of plasma ACTH (baseline: 106.4+/-48.6; vs. just after stress: 388.9+/-56.1 pg/ml, P<0.05) and B (baseline: 198.6+/-46.8 vs. 15 min after stress: 378.5+/-13.6 ng/ml, P<0.05) in the control group. Plasma OT tended to increase after stress, although the change was not significantly different (baseline: 29.8+/-6.5; just after stress: 65.6+/-18.2 pg/ml). I.c.v. injection of AM at 3 min before the stress did not significantly affect stress-induced changes of plasma ACTH, B and OT. These results suggest that AM has an inhibitory effect on OT-induced ACTH release in vitro and the inhibitory effect may be overwhelmed in ACTH and B response to shaker stress.