Hiroaki Fujihara

Central control of fever and female body temperature by RANKL/RANK

Receptor-activator of NF-κB ligand (TNFSF11, also known as RANKL, OPGL, TRANCE and ODF) and its tumour necrosis factor (TNF)-family receptor RANK are essential regulators of bone remodelling, lymph node organogenesis and formation of a lactating mammary gland. RANKL and RANK are also expressed in the central nervous system. However, the functional relevance of RANKL/RANK in the brain was entirely unknown. Here we report that RANKL and RANK have an essential role in the brain. In both mice and rats, central RANKL injections trigger severe fever. Using tissue-specific Nestin-Cre and GFAP-Cre rankfloxed deleter mice, the function of RANK in the fever response was genetically mapped to astrocytes. Importantly, Nestin-Cre and GFAP-Cre rankfloxed deleter mice are resistant to lipopolysaccharide-induced fever as well as fever in response to the key inflammatory cytokines IL-1β and TNFα. Mechanistically, RANKL activates brain regions involved in thermoregulation and induces fever via the COX2-PGE2/EP3R pathway. Moreover, female Nestin-Cre and GFAP-Cre rankfloxed mice exhibit increased basal body temperatures, suggesting that RANKL and RANK control thermoregulation during normal female physiology. We also show that two children with RANK mutations exhibit impaired fever during pneumonia. These data identify an entirely novel and unexpected function for the key osteoclast differentiation factors RANKL/RANK in female thermoregulation and the central fever response in inflammation.

Oxytocin: Crossing the Bridge between Basic Science and Pharmacotherapy

Is oxytocin the hormone of happiness? Probably not. However, this small nine amino acid peptide is involved in a wide variety of physiological and pathological functions such as sexual activity, penile erection, ejaculation, pregnancy, uterus contraction, milk ejection, maternal behavior, osteoporosis, diabetes, cancer, social bonding, and stress, which makes oxytocin and its receptor potential candidates as targets for drug therapy. In this review, we address the issues of drug design and specificity and focus our discussion on recent findings on oxytocin and its heterotrimeric G protein‐coupled receptor OTR. In this regard, we will highlight the following topics: (i) the role of oxytocin in behavior and affectivity, (ii) the relationship between oxytocin and stress with emphasis on the hypothalamo–pituitary–adrenal axis, (iii) the involvement of oxytocin in pain regulation and nociception, (iv) the specific action mechanisms of oxytocin on intracellular Ca2+ in the hypothalamo neurohypophysial system (HNS) cell bodies, (v) newly generated transgenic rats tagged by a visible fluorescent protein to study the physiology of vasopressin and oxytocin, and (vi) the action of the neurohypophysial hormone outside the central nervous system, including the myometrium, heart and peripheral nervous system. As a short nine amino acid peptide, closely related to its partner peptide vasopressin, oxytocin appears to be ideal for the design of agonists and antagonists of its receptor. In addition, not only the hormone itself and its binding to OTR, but also its synthesis, storage and release can be endogenously and exogenously regulated to counteract pathophysiological states. Understanding the fundamental physiopharmacology of the effects of oxytocin is an important and necessary approach for developing a potential pharmacotherapy.

PKCλ regulates glucose-induced insulin secretion through modulation of gene expression in pancreatic β cells

Altered regulation of insulin secretion by glucose is characteristic of individuals with type 2 diabetes mellitus, although the mechanisms that underlie this change remain unclear. We have now generated mice that lack the lambda isoform of PKC in pancreatic beta cells (betaPKClambda(-/-) mice) and show that these animals manifest impaired glucose tolerance and hypoinsulinemia. Furthermore, insulin secretion in response to high concentrations of glucose was impaired, whereas the basal rate of insulin release was increased, in islets isolated from betaPKClambda(-/-) mice. Neither the beta cell mass nor the islet insulin content of betaPKClambda(-/-) mice differed from that of control mice, however. The abundance of mRNAs for Glut2 and HNF3beta was reduced in islets of betaPKClambda(-/-) mice, and the expression of genes regulated by HNF3beta was also affected (that of Sur1 and Kir6.2 genes was reduced, whereas that of hexokinase 1 and hexokinase 2 genes was increased). Normalization of HNF3beta expression by infection of islets from betaPKClambda(-/-) mice with an adenoviral vector significantly reversed the defect in glucose-stimulated insulin secretion. These results indicate that PKClambda plays a prominent role in regulation of glucose-induced insulin secretion by modulating the expression of genes important for beta cell function.

Exaggerated Response of Arginine Vasopressin-Enhanced Green Fluorescent Protein Fusion Gene to Salt Loading without Disturbance of Body Fluid Homeostasis in Rats

We examined the effects of chronic salt loading on the hypothalamic expressions of the enhanced green fluorescent protein (eGFP), arginine vasopressin (AVP) and oxytocin (OXT) genes in AVP‐eGFP transgenic rats that expressed eGFP in the hypothalamic AVP‐containing neurones. In these rats, salt loading for 5 days caused a marked increase of the eGFP fluorescence in the magnocellular divisions of the paraventricular nucleus (PVN), the supraoptic nucleus (SON) and the internal layer of the median eminence. Expression of the eGFP gene was increased seven‐ to eight‐fold in the PVN and SON of salt‐loaded rats in comparison with euhydrated rats. By contrast, none of these changes were observed in the suprachiasmatic nucleus. The expression of the AVP and OXT genes was increased 1.5‐ to two‐fold in the PVN and SON of salt‐loaded nontransgenic (control) and transgenic rats. There were no differences in the expression levels of the AVP and OXT genes in the PVN and SON between nontransgenic (control) and transgenic animals under normal conditions and after salt loading. In the posterior pituitary gland, the intensity of the eGFP fluorescence did not change after salt loading for 5 days, but increased after 10 days of salt loading. Upon salt loading, significant increases in the plasma AVP concentrations, plasma osmolality and plasma Na+ were observed. Furthermore, there were no significant differences in changes of water intake, food intake, urine volume, urine osmolality, urine Na+ concentrations, and the body weights in both models under normal or salt‐loaded conditions. Our results show that the response of the AVP‐eGFP fusion gene to chronic salt loading is exaggerated, and humoral responses such as AVP and OXT and the body fluid homeostasis are maintained in AVP‐eGFP transgenic rats. The AVP‐eGFP transgenic rat gives us a new opportunity to study the dynamics of the AVP system in vivo.

Short-term sleep disturbance enhances brain-derived neurotrophic factor gene expression in rat hippocampus by acting as internal stressor

Rats were subjected to nonselective sleep disturbance for short periods under conditions designed to minimize the adverse influence of external stresses, such as environmental conditions and restricted movement, and both brain-derived neurotrophic factor (BDNF) protein and its mRNA levels in the brain were then determined to investigate the influence of sleep disturbance itself on BDNF gene expression. Total sleep duration was partially but significantly reduced by disturbing the sleep/wake cycle for 1 and 2 h, gradually increased according to the time of disturbance, then returned to control levels at 6 h after the beginning of sleep disturbance. Under these conditions, the slight but significant elevation of corticotrophin-releasing factor (CRF) mRNA levels in the paraventricular nucleus (PVN) was observed at an early stage of the sleep disturbance period. Sleep disturbance induced the elevation of both BDNF protein and its mRNA levels in the hippocampus but not in the cerebellum or the brainstem, and the elevated BDNF mRNA expression in the hippocampus returned toward basal levels during the sleep recovery period when the rebound of sleep duration was observed. These findings suggest the possibility that short-term disturbance of the sleep/wake cycle and, hence, the partial reduction of non-REM sleep duration, might exert a potential influence on neuronal and/or glial cells as an internal stressor, resulting in the elevation of BDNF gene expression in rat hippocampus.

Physiological Studies of Stress Responses in the Hypothalamus of Vasopressin-Enhanced Green Fluorescent Protein Transgenic Rat

Arginine vasopressin (AVP) plays an important role in stress‐induced activation of the hypothalamic‐pituitary adrenal axis. In the present study, AVP‐enhanced green fluorescent protein (eGFP) transgenic rats were used to investigate changes in AVP‐eGFP expression in the hypothalamic paraventricular nucleus (PVN) and the median eminence (ME) upon exposure to stress conditions. The eGFP fluorescence in the parvocellular division of the PVN (pPVN) was markedly increased 5 days after bilateral adrenalectomy (ADX) and it was colocalised with corticotrophin‐releasing hormone‐like immunoreactivity in the pPVN. Peripheral administration of dexamethasone completely suppressed the increase of eGFP fluorescence in the pPVN and the external layer of the ME (eME) after bilateral ADX. Significant increases of eGFP fluorescence were observed in the pPVN 6, 12, 24 and 48 h after intraperitoneal (i.p.) administration of lipopolysaccharide (LPS). In the eME, eGFP fluorescence was significantly increased 48 h after i.p. administration of LPS. By contrast, eGFP fluorescence changed neither in the magnocellular division of the PVN, nor the internal layer of the ME after i.p. administration of LPS. Our results indicate that AVP‐eGFP transgenic rats are useful animal model to study dynamic changes of AVP expression in the hypothalamus under stressful conditions.

Single eight-hour shift of light-dark cycle increases brain-derived neurotrophic factor protein levels in the rat hippocampus.

We previously reported that an eight hour phase advance in the light-dark (LD) cycle increases sleep in rats. Brain-derived neurotrophic factor (BDNF) is suggested to be one of the sleep and circadian regulating factors. We have therefore observed the responses of BDNF protein in the hippocampus, cerebellum and brainstem under conditions of LD change. BDNF protein was quantitatively measured using an ELISA kit. Under an 8-h LD phase advance, the levels of hippocampal BDNF were significantly increased on the day of the phase change, while the levels in the cerebellum and brainstem remained constant. Plasma corticosterone levels were not largely affected. Thus, a single LD shift acutely affects hippocampal BDNF metabolism with no large stress response.

Centrally administered relaxin-3 induces Fos expression in the osmosensitive areas in rat brain and facilitates water intake.

The expression of the relaxin-3 gene, detected as a new member of the insulin superfamily using human genomic databases, is abundantly present in the brain and testis. Intracerebroventricularly (icv) administered relaxin-3 stimulates food intake. Icv administered relaxin (identical to relaxin-2 in humans) affects the secretion of vasopressin and drinking behavior. Relaxin-3 partly binds relaxin family peptide receptor 1, which is a specific receptor to relaxin. Thus, we hypothesized that relaxin-3 would have physiological effects in the body fluid balance. However, the effects of relaxin-3 in the body fluid balance remain unknown. In the present study, we revealed that icv administered relaxin-3 induced dense Fos-like immunoreactivity (Fos-LI) in the rat hypothalamus and circumventricular organs including the organum vasculosum of the lamina terminalis, the median preoptic nucleus, supraoptic nucleus (SON), the subfornical organ (SFO) and the paraventricular nucleus (PVN), that are related to the central regulation of body fluid balance. Icv administered relaxin-3 (54, 180 and 540 pmol/rat) also induced a significant increase in c-fos gene expression in a dose-dependent manner in the SON, SFO and PVN. Further, icv administered relaxin-3 (180 pmol/rat) significantly increased water intake, and the effect was as strong as that of relaxin-2 (180 pmol/rat). These results suggest that icv administered relaxin-3 activates osmosensitive areas in the brain and plays an important role in the regulation of body fluid balance.

Chronic Osmotic Stimuli Increase Salusin-β-Like Immunoreactivity in the Rat Hypothalamo-Neurohypophyseal System : Possible Involvement of Salusin-β on [Ca2+]i Increase and Neurohypophyseal Hormone Release from the Axon Terminals

Salusin‐α and ‐β were recently discovered as bioactive endogenous peptides. In the present study, we investigated the effects of chronic osmotic stimuli on salusin‐β‐like immunoreactivity (LI) in the rat hypothalamo‐neurohypophyseal system. We examined the effects of salusin‐β on synaptic inputs to the rat magnocellular neurosecretory cells (MNCs) of the supraoptic nucleus (SON) and neurohypophyseal hormone release from both freshly dissociated SONs and neurohypophyses in rats. Immunohistochemical studies revealed that salusin‐β‐LI neurones and fibres were markedly increased in the SON and the magnocellular division of the paraventricular nucleus after chronic osmotic stimuli resulting from salt loading for 5 days and dehydration for 3 days. Salusin‐β‐LI fibres and varicosities in the internal zone of the median eminence and the neurohypophysis were also increased after osmotic stimuli. Whole‐cell patch‐clamp recordings from rat SON slice preparations showed that salusin‐β did not cause significant changes in the excitatory and inhibitory postsynaptic currents of the MNCs. In vitro hormone release studies showed that salusin‐β evoked both arginine vasopressin (AVP) and oxytocin release from the neurohypophysis, but not the SON. In our hands, in the neurohypophysis, a significant release of AVP and oxytocin was observed only at concentrations from 100 nm and above of salusin‐β. Low concentrations below 100 nm were ineffective both on AVP and oxytocin release. We also measured intracellular calcium ([Ca2+]i) increase induced by salusin‐β on freshly‐isolated single nerve terminals from the neurohypophysis devoid of pars intermedia. Furthermore, this salusin‐β‐induced [Ca2+]i increase was blocked in the presence of high voltage activated Ca2+channel blockers. Our results suggest that salusin‐β may be involved in the regulation of body fluid balance by stimulating neurohypophyseal hormone release from nerve endings by an autocrine/paracrine mechanism.

Parathyroid Hormone-Related Protein Induces Cachectic Syndromes without Directly Modulating the Expression of Hypothalamic Feeding-Regulating Peptides

Purpose: Parathyroid hormone-related protein (PTHrP) is a causative factor of humoral hypercalcemia of malignancy (HHM) and concurrent anorexia and wasting. Because changes in the expression of hypothalamic feeding-regulating peptides can directly affect appetites and thereby can cause anorexia and wasting, we addressed whether the cachectic syndromes induced by PTHrP rely on the action of hypothalamic feeding-regulating peptides. Experimental Design: Rats were inoculated with a LC-6 human cancer xenograft that secreted PTHrP, and the mRNA levels of the hypothalamic feeding-regulating peptide genes and serum leptin levels were examined before and after the development of HHM by in situ hybridization histochemistry and ELISA, respectively. Some rats were given the anti-PTHrP antibody. Results and Conclusion: The mRNA levels for the orexigenic peptides, such as the agouti-related protein and the neuropeptide Y in the arcuate nucleus (Arc), were significantly increased after the development of HHM, whereas the mRNA levels for the anorexigenic peptides, such as the proopiomelanocortin in the Arc, the cocaine and amphetamine-regulated transcript in the Arc, and the corticotropin-releasing factor in the paraventricular nucleus, were significantly decreased after the development of HHM. Plasma leptin levels were also reduced in cachectic rats, and the administration of anti-PTHrP antibody to the cachectic rats not only improved the cachectic symptoms but also restored the mRNA levels of these orexigenic and anorexigenic peptides, except for orexin. Thus, PTHrP induces HHM and concurrent cachectic syndromes by mechanisms other than directly modulating the leptin or hypothalamic feeding-regulated peptides.