Stress-Induced Glucocorticoid Release Upregulates Uncoupling Protein-2 Expression and Enhances Resistance to Endotoxin-Induced Lethality.

OBJECTIVE: Although psychological and/or physiological stress has been well documented to influence immune responses, the precise mechanism for immunomodulation remains to be elucidated. The present work describes the role of the hypothalamic-pituitary-adrenal (HPA) axis in the mechanism of stress-mediated enhanced-resistance to lethality after lipopolysaccharide (LPS) injection. METHODS/RESULTS: Preconditioning with restraint stress (RS) resulted in enhanced activation of the HPA axis in response to LPS injection and suppressed LPS-induced release of proinflammatory cytokines and nitric oxide metabolites. Melanocortin 2 receptor-deficient mice (MC2R(-/-)) failed to increase plasma levels of glucocorticoids in response to LPS injection, and exhibited high sensitivity to LPS-induced lethality with enhanced release of proinflammatory cytokines as compared with MC2R(+/-) mice. Real-time PCR analysis revealed that RS induced upregulation of uncoupling protein-2 (UCP2) in macrophages in the lung and the liver of MC2R(+/-), but not of MC2R(-/-), mice. In addition, RS increased UCP2-dependent uncoupling activity of isolated mitochondria from the liver of MC2R(+/-), but not of MC2R(-/-), mice. In vitro study revealed that corticosterone and dexamethasone directly increased UCP2 expression in mouse RAW 264.7 macrophages and suppressed the generation of LPS-induced mitochondrial reactive oxygen species (ROS) and TNF-α production. Knockdown of UCP2 by small interfering RNA blunted the dexamethasone action for suppressing LPS-induced mitochondrial ROS and TNF-α production. CONCLUSION: The present work suggests that RS enhances activation of the HPA axis to release glucocorticoids and upregulation of UCP2 in macrophages, thereby increasing the resistance to endotoxin-induced systemic inflammation and death.

Notch Signaling-Modified Mesenchymal Stem Cell Patch Improves Left Ventricular Function via Arteriogenesis Induction in a Rat Myocardial Infarction Model.

For ischemic cardiomyopathy (ICM) with limited therapeutic options, the induction of arteriogenesis has the potential to improve cardiac function through major restoration of blood flow. We hypothesized that transplantation of a Notch signaling-modified mesenchymal stem cell (SB623 cell) patch would induce angiogenesis and arteriogenesis in ischemic lesions, leading to improvement of left ventricular (LV) function in a rat ICM model. Two weeks after the induction of ischemia, SB623 cell patch transplantation into ICM rats (SB group, n = 10) or a sham operation (no-treatment group, n = 10) was performed. The LV ejection fraction was significantly improved at 6 weeks after SB623 cell patch transplantation (P < 0.001). Histological findings revealed that the number of von Willebrand factor (vWF)-positive capillary vessels (P < 0.01) and alpha smooth muscle actin (αSMA)- and vWF-positive arterioles with a diameter greater than 20 µm (P = 0.002) was significantly increased in the SB group, suggesting the induction of angiogenesis and arteriogenesis. Moreover, rat cardiomyocytes treated with SB623 cell patch transplantation showed upregulation of ephrin-B2 (P = 0.03) and EphB4 (P = 0.01) gene expression, indicating arteriogenesis induction. In conclusion, SB623 cell patch transplantation improved LV function by inducing angiogenesis and arteriogenesis in a rat ICM model.

Cell therapies for acute and chronic traumatic brain injury.

Traumatic brain injury (TBI) is a global health problem, for which there are no approved therapies. Advances in acute clinical care have improved post-TBI survival, yet many patients are left with chronic TBI-related disabilities (i.e. chronic TBI). Existing treatments that focus on rehabilitation and symptom management do not modify the disease and have limited effectiveness. Consequently, chronic TBI-related disabilities remain a significant unmet medical need. Cell therapies have neuroprotective and neurorestorative effects which are believed to modify the disease. In this article, we review the safety and efficacy of cell therapies in early-phase clinical studies that have shown potential to improve outcomes in acute to chronic phases of TBI.

RhoH plays critical roles in Fc epsilon RI-dependent signal transduction in mast cells.

RhoH is an atypical small G protein with defective GTPase activity that is specifically expressed in hematopoietic lineage cells. RhoH has been implicated in regulation of several physiological processes including hematopoiesis, integrin activation, and T cell differentiation and activation. In the present study, we investigated the role of RhoH in mast cells by generating RhoH knockout mice. Despite observing normal development of mast cells in vivo, passive systemic anaphylaxis and histamine release were impaired in these mice. We also observed defective degranulation and cytokine production upon FcepsilonRI ligation in RhoH-deficient bone marrow-derived mast cells. Furthermore, FcepsilonRI-dependent activation of Syk and phosphorylation of its downstream targets, including LAT, SLP76, PLCgamma1, and PLCgamma2 were impaired, however phosphorylation of the gamma-subunit of FcepsilonRI remained intact. We also found RhoH-Syk association that was greatly enhanced by active Fyn. Our results indicate that RhoH regulates FcepsilonRI signaling in mast cells by facilitating Syk activation, possibly as an adaptor molecule for Syk.

Notch signaling-modified mesenchymal stem cells improve tissue perfusion by induction of arteriogenesis in a rat hindlimb ischemia model.

Notch signaling-modified human mesenchymal stem cell, SB623 cell, is a promising cell therapy product for ischemic stroke. With the aim to expand indications for their use for critical limb-threatening ischemia (CLTI), we hypothesized that SB623 cells improved tissue perfusion by inducing angiogenesis or arteriogenesis in a hindlimb ischemia model rat. In Sprague-Dawley rats, hindlimb ischemia was generated by femoral artery removal, then seven days after ischemic induction 1 × 105 SB623 cells or PBS was injected into the ischemic adductor muscle. As compared with the PBS group, tissue perfusion was significantly increased in the SB623 group. While capillary density did not vary between the groups, αSMA- and vWF-positive arterioles with a diameter > 15 µm were significantly increased in the SB623 group. Whole transcriptome analysis of endothelial cells co-cultured with SB623 cells showed upregulation of the Notch signaling pathway as well as several other pathways potentially leading to arteriogenesis. Furthermore, rat muscle treated with SB623 cells showed a trend for higher ephrin-B2 and significantly higher EphB4 expression, which are known as arteriogenic markers. In the hindlimb ischemia model, SB623 cells improved tissue perfusion by inducing arteriogenesis, suggesting a promising cell source for treatment of CLTI.

Cell Therapy for Chronic TBI: Interim Analysis of the Randomized Controlled STEMTRA Trial.

OBJECTIVE: To determine if chronic motor deficits secondary to traumatic brain injury (TBI) can be improved by implantation of allogeneic modified bone marrow-derived mesenchymal stromal/stem cells (SB623). METHODS: This 6-month interim analysis of the 1-year double-blind, randomized, surgical sham-controlled, phase 2 STEMTRA trial (NCT02416492) evaluated safety and efficacy of the stereotactic intracranial implantation of SB623 in patients with stable chronic motor deficits secondary to TBI. Patients in this multi-center trial (N = 63) underwent randomization in a 1:1:1:1 ratio to 2.5 × 106, 5.0 × 106, 10 × 106 SB623 cells or control. Safety was assessed in patients who underwent surgery (N = 61), and efficacy in the modified intent-to-treat population of randomized patients who underwent surgery (N = 61; SB623 = 46, control = 15). RESULTS: The primary efficacy endpoint of significant improvement from baseline of Fugl-Meyer Motor Scale score at 6 months for SB623-treated patients was achieved. SB623-treated patients improved by (LS mean [SE]) +8.3 (1.4) vs +2.3 (2.5) for control at 6 months, the LS mean difference was 6.0 (95% CI: 0.3-11.8); p = 0.040. Secondary efficacy endpoints improved from baseline, but were not statistically significant vs control at 6 months. There were no dose-limiting toxicities or deaths, and 100% of SB623-treated patients experienced treatment-emergent adverse events vs 93.3% of control patients (p = 0.25). CONCLUSIONS: SB623 cell implantation appeared to be safe and well tolerated, and patients implanted with SB623 experienced significant improvement from baseline motor status at 6 months compared to controls. CLASSIFICATION OF EVIDENCE: This study provides Class I evidence that implantation of SB623 was well tolerated and associated with improvement in motor status.

The role of endogenous glucocorticoids in lymphocyte development in melanocortin receptor 2-deficient mice.

Glucocorticoids are extensively used in anti-inflammatory therapy and are thought to contribute to the steady-state regulation of hematopoiesis and lymphopoiesis. We have previously established MC2R(-/-) mice, a model of familial glucocorticoid deficiency, that show several similarities to patients with this disease, including undetectable levels of corticosterone, despite high levels of ACTH and unresponsiveness to ACTH. In this study, we analyzed the possible roles of endogenous glucocorticoids in hematopoiesis and lymphopoiesis in MC2R(-/-) and CRH(-/-) mice as models of chronic adrenal insufficiency. Our analysis of total peripheral blood cell counts revealed that the number of lymphocytes was increased and the number of erythrocytes was slightly, but significantly, decreased in MC2R(-/-) mice. Numbers of immature double negative (CD4(-) CD8(-)) thymocytes, transitional type 1 B cells in the spleen, and pre-B cells in the bone marrow, were significantly increased in MC2R(-/-) mice, suggesting that endogenous glucocorticoids contribute to steady-state regulation of lymphopoiesis. Oral glucocorticoid supplementation reversed peripheral blood cell counts and reduced numbers of T and B cells in the thymus and the spleen. T cells in the thymus and B cells in the spleen were also increased in CRH(-/-) mice, another animal model of chronic adrenal insufficiency. MC2R(-/-) mice were sensitive to age-related thymic involution, but they were resistant to fasting-associated thymic involution. Our data support the idea that endogenous glucocorticoids contribute to stress-induced as well as steady-state regulation of hematopoiesis and lymphopoiesis.

Melanocortin 2 receptor is required for adrenal gland development, steroidogenesis, and neonatal gluconeogenesis.

ACTH (i.e., corticotropin) is the principal regulator of the hypothalamus-pituitary-adrenal axis and stimulates steroidogenesis in the adrenal gland via the specific cell-surface melanocortin 2 receptor (MC2R). Here, we generated mice with an inactivation mutation of the MC2R gene to elucidate the roles of MC2R in adrenal development, steroidogenesis, and carbohydrate metabolism. These mice, the last of the knockout (KO) mice to be generated for melanocortin family receptors, provide the opportunity to compare the phenotype of proopiomelanocortin KO mice with that of MC1R-MC5R KO mice. We found that the MC2R KO mutation led to neonatal lethality in three-quarters of the mice, possibly as a result of hypoglycemia. Those surviving to adulthood exhibited macroscopically detectable adrenal glands with markedly atrophied zona fasciculata, whereas the zona glomerulosa and the medulla remained fairly intact. Mutations of MC2R have been reported to be responsible for 25% of familial glucocorticoid deficiency (FGD) cases. Adult MC2R KO mice resembled FGD patients in several aspects, such as undetectable levels of corticosterone despite high levels of ACTH, unresponsiveness to ACTH, and hypoglycemia after prolonged (36 h) fasting. However, MC2R KO mice differ from patients with MC2R-null mutations in several aspects, such as low aldosterone levels and unaltered body length. These results indicate that MC2R is required for postnatal adrenal development and adrenal steroidogenesis and that MC2R KO mice provide a useful animal model by which to study FGD.

Bone phenotype in melanocortin 2 receptor-deficient mice.

Considering that stress condition associated with osteoporosis, the hypothalamic-pituitary-adrenal (HPA) axis, which is essential for central stress response system, is implicated in regulating bone mass accrual. Melanocortin 2 receptor (MC2R), the receptor of adrenocorticotropic hormone is expressed in both adrenal gland cells and bone cells. To elucidate the role of HPA axis in bone metabolism, we assessed the skeletal phenotype of MC2R deficient mice (MC2R -/- mice). We first examined bone mineral density and cortical thickness of femur using dual x-ray absorptiometry and micro-computed tomography. We then conducted histomorphometric analysis to calculate the static and dynamic parameters of vertebrae in MC2R -/- mice. The levels of osteoblastic marker genes were examined by quantitative PCR in primary osteoblasts derived from MC2R -/- mice. Based on these observations, bone mineral density of femur in MC2R -/- mice was increasing relative to litter controls. Meanwhile, the thickness of cortical bone of femur in MC2R -/- mice was remarkably elevated. Moreover, serum osteocalcin level was drastically raised in MC2R -/- mice. However, bone histomorphometry revealed that static and dynamic parameters reflecting bone formation and resorption were unchanged in vertebrae of MC2R -/- mice compared to the control, indicating that MC2R function may be specific to appendicular bone than axis bone. Taken together, the HPA axis due to deletion of MC2R is involved in bone metabolism.

Characterization of mice deficient in melanocortin 2 receptor on a B6/Balbc mix background.

We have previously reported that Melanocortin 2 receptor (MC2R(-/-)) deficient mice on B6 N5 generations exhibited macroscopically detectable adrenal glands with markedly atrophied zona fasciculata (zF) and lack of detectable levels of corticosterone, and reduced serum concentrations of aldosterone and epinephrine. All MC2R(-/-) mice on B6/N8 background die within 2 days after birth, while about half of the MC2R(-/-) mice on B6/Balbc mix background survived to adulthood. Both male and female MC2R(-/-) mice were fertile, suggesting that normal development and function of reproductive organs. MC2R(-/-) mice delivered from MC2R(-/-) dams failed to survive due to lung failure, suggesting that fetal or maternal corticosterone is essential for lung maturation. MC2R(-/-) mice failed to activate the hypothalamic-pituitary-adrenal axis in response to both immune and non-immune stimuli. MC2R(-/-) mice maintained glomerular structure and achieved electrolyte homeostasis by the activation of the renin-angiotensin-aldosterone system under low aldosterone and undetectable levels of corticosterone.

Combined interleukin-6 and interleukin-1 deficiency causes obesity in young mice.

Proinflammatory cytokines including interleukin (IL)-1 and IL-6 exert pleiotropic effects on the neuro-immuno-endocrine system. Previously, we showed that IL-1 receptor antagonist-deficient (IL-1Ra(-/-)) mice show a lean phenotype due to an abnormal lipid metabolism. On the contrary, it was reported that IL-6(-/-) mice exhibit obesity after 6 months of age. This study sought to assess the roles of IL-1 and IL-6 in body weight homeostasis. We generated mice deficient in IL-6 and IL-1Ra (IL-6(-/-) IL-1Ra(-/-)) and IL-6, IL-1alpha, and IL-1beta (IL-6(-/-) IL-1(-/-)). IL-6(-/-) IL-1Ra(-/-) mice exhibited a lean phenotype, similar to IL-1Ra(-/-) mice. On the other hand, IL-6(-/-) IL-1(-/-) mice became obese as early as 10 weeks of age, while IL-1(-/-) mice and IL-6(-/-) mice were normal at this age. The daily food intake was significantly higher in IL-6(-/-) IL-1(-/-) mice than in IL-6(-/-) IL-1(+/-) mice, while energy expenditure was comparable in these two strains. Acute anorexia induced by peripheral administration of IL-1 was significantly suppressed in IL-6(-/-) IL-1(-/-) mice, but not in IL-1(-/-) mice or IL-6(-/-) mice compared with wild-type mice. These results indicate that IL-1 and IL-6 are both involved in the regulation of body fat in a redundant manner in young mice.

Peripheral TNFalpha, but not peripheral IL-1, requires endogenous IL-1 or TNFalpha induction in the brain for the febrile response.

It is known that peripherally administered IL-1 and TNFalpha induce fever through mechanisms involving prostaglandin (PG)E2. In this report, we compared the signaling cascade induced in the brain by TNFalpha and IL-1. Peripheral administration of TNFalpha-induced enhanced fever in IL-1 Receptor antagonist KO mice, suggesting that IL-1 is involved in the TNFalpha mediated fever. IL-1alpha, but not TNFalpha, induced fever in IL-1alpha/beta/TNFalpha KO mice, although central administration of TNFalpha-induced fever. Only IL-1alpha, but not TNFalpha, induced IL-6 in the IL-1alpha/beta/TNFalpha KO mouse brain, while both cytokines induced cyclooxygenase (Cox)-2. I.c.v. administration of PGE2 induced only transient fever in contrast to the TNFalpha- or IL-1alpha-induced fever that lasted longer. Taken together, either IL-1 or TNFalpha induction in the brain is required for the response induced by TNFalpha but not by IL-1alpha, and that both Cox-2 and IL-6 induction are required for prolonged febrile response against these cytokines.

Basal Insulin Persistence, Associated Factors, and Outcomes After Treatment Initiation: A Retrospective Database Study Among People with Type 2 Diabetes Mellitus in Japan.

INTRODUCTION: The objective of this study was to assess basal insulin persistence, associated factors, and economic outcomes for insulin-naïve people with type 2 diabetes mellitus (T2DM) in Japan. METHODS: People aged at least 18 years with T2DM with first claim for basal insulin between May 2006 and April 2013 (index date), no insulin use before index date, and continuous insurance coverage for 6 months before (baseline) and 12 months after index date were selected from the Japan Medical Center Database. On the basis of whether there were at least 30-day gaps in basal insulin treatment, patients were classified as continuers (no gap), interrupters (at least one prescription after gap), and discontinuers (no prescription after gap). A multinomial logistic regression model identified factors associated with persistence. Annual healthcare resource use and costs in the year after initiation were compared between continuers and interrupters and between continuers and discontinuers using propensity score-based inverse probability weighting to adjust for baseline differences. RESULTS: Of the 827 people included (mean age 50 years, ca. 71% male), 36% continued, 42% interrupted, and 22% discontinued basal insulin therapy in the year after initiation. Having at least one inpatient visit and using fewer classes of non-insulin antihyperglycemic medications during baseline were associated with lower likelihoods of continuing therapy. Relative to interrupters and discontinuers, continuers had lower hospitalization rates [continuers, 12.7%; interrupters, 25.4% (p < 0.001); discontinuers, 28.4% (p < 0.001)] and lower inpatient costs [continuers, ¥132,013; interrupters, ¥225,745 (p = 0.054); discontinuers, ¥320,582 (p = 0.036)], but higher pharmacy costs [continuers, ¥158,403; interrupters, ¥134,301 (p = 0.039); discontinuers, ¥121,593 (p = 0.002)] in the year after insulin initiation. Total healthcare costs were similar for the three cohorts. CONCLUSIONS: Substantial proportions of people with T2DM in Japan interrupt or discontinue basal insulin within the year after initiation, and they have higher rates and costs of hospitalizations than patients who continue with their insulin therapy. Further research is needed to understand reasons behind basal insulin persistence and the implications thereof to help clinicians manage T2DM more effectively. FUNDING: Eli Lilly and Company, Boehringer Ingelheim.

Involvement of corticotropin-releasing hormone- and interleukin (IL)-6-dependent proopiomelanocortin induction in the anterior pituitary during hypothalamic-pituitary-adrenal axis activation by IL-1alpha.

IL-1alpha/beta and IL-6 are endogenous modulator of hypothalamo-pituitary-adrenal axis (HPAA) and are thought to play key roles in immune-neuroendocrine interactions during inflammation. Here, we show IL-1alpha induced a normal HPAA activation in IL-1alpha/beta knockout (KO) and IL-6 KO mice at 1 h; however, at 6 h HPAA activation was reduced relative to wild-type mice, indicating a role for endogenous IL-1alpha/beta and IL-6 in prolonged HPAA activation. We found that the induction of proopiomelanocortin (POMC) transcript in the anterior pituitary (AP) at 6 h in response to IL-1alpha was reduced in IL-1alpha/beta KO and IL-6 KO mice, as well as in CRH KO mice, suggesting IL-1alpha/beta, IL-6, and CRH are all required for POMC induction. The induction of CRH transcript in the paraventricular nucleus at 6 h and plasma IL-6 levels, in response to IL-1alpha, were reduced in IL-1alpha/beta KO mice. Because IL-1alpha-induced activation of signal transducer and activator of transcription 3 in the AP was also suppressed in IL-6 KO mice, we suggest that plasma IL-6 is first induced by IL-1alpha, and IL-6 activates signal transducer and activator of transcription 3 in the AP, leading to the induction of POMC in concert with CRH. Our results suggest a role for IL-1alpha/beta in the induction of POMC in the AP through the induction of two independent pathways, CRH and IL-6.

Interleukin (IL)-6, but not IL-1, induction in the brain downstream of cyclooxygenase-2 is essential for the induction of febrile response against peripheral IL-1alpha.

IL-1 is an endogenous pyrogen produced upon inflammation or infection. Previously, we showed that, upon injection with turpentine, IL-1 is induced in the brain in association with the development of fever. The role of endogenous IL-1 in the brain and the signaling cascade to activate thermosensitive neurons, however, remain to be elucidated. In this report, febrile response was analyzed after peripheral injection of IL-1alpha. We found that a normal febrile response was induced even in IL-1alpha/beta-deficient mice, indicating that production of IL-1 in the brain is not necessarily required for the response. In contrast, IL-6-deficient mice did not exhibit a febrile response. Cyclooxygenase (Cox)-2 expression in the brain was strongly induced 1.5 h after injection of IL-1alpha, whereas IL-6 expression was observed 3 h after the injection. Cox-2 expression in the brain was not influenced by IL-6 deficiency, whereas indomethacin, an inhibitor of cyclooxygenases, completely inhibited induction of IL-6. These observations suggest a mechanism of IL-1-induced febrile response in which IL-1 in the blood activates Cox-2, with the resulting prostaglandin E(2) inducing IL-6 in the brain, leading to the development of fever.

Expression and regulation of neuromedin B in pituitary corticotrophs of male melanocortin 2 receptor-deficient mice.

The hypothalamic-pituitary-adrenal (HPA) axis is a major part of the neuroendocrine system that controls responses to stress, and has an important function in the regulation of various body processes. We previously created a mouse line deficient in the melanocortin 2 receptor (MC2R). MC2R-deficient mice (MC2R(-/-) mice) have high adrenocorticotropic hormone (ACTH) levels because of undetectable corticosterone levels. Increased neuromedin B (NMB) expression was recently reported in the pituitary gland of adrenalectomized mice, a model for acute adrenal insufficiency. To investigate gene expression in the pituitary gland under chronic adrenal deficiency, we examined the pituitary gland of MC2R(-/-) mice, a model of chronic adrenal insufficiency. To understand the molecular background of pituitary cells under chronic adrenal deficiency, we first performed DNA microarray analyses using the pituitary glands of the MC2R(-/-) mice. The DNA microarray analysis and real-time polymerase chain reaction showed that NMB expression was higher in the MC2R(-/-) than in the wild-type (WT) mice. We detected NMB expression in the MC2R(-/-) pituitary corticotrophs by immunohistochemistry using the specific antibodies for ACTH and NMB. In addition, the plasma NMB concentration was significantly higher in the MC2R(-/-) mice than in the WT mice. Subcutaneous implantation of a sustained-release corticosterone pellet decreased the expression of NMB mRNA as well as pituitary proopiomelanocortin mRNA. In isolated anterior pituitary cells, NMB mRNA expression was increased by the administration of corticotropin-releasing hormone (CRH) and was suppressed by dexamethasone treatment. In this study, we first demonstrate NMB expression in corticotrophs and its regulation by CRH and glucocorticoids. Furthermore, corticotrophs seemed to secrete NMB into the systemic circulation.

Netrin-4 derived from murine vascular endothelial cells inhibits osteoclast differentiation in vitro and prevents bone loss in vivo.

Bone is a highly vascularized organ, thus angiogenesis is a vital process during bone remodeling. However, the role of vascular systems in bone remodeling is not well recognized. Here we show that netrin-4 inhibits osteoclast differentiation in vitro and in vivo. Co-cultures of bone marrow macrophages with vascular endothelial cells markedly inhibited osteoclast differentiation. Adding a neutralizing antibody, or RNA interference against netrin-4, restored in vitro osteoclast differentiation. Administration of netrin-4 prevented bone loss in an osteoporosis mouse model by decreasing the osteoclast number. We propose that vascular endothelial cells interact with bone in suppressing bone through netrin-4.

The role of glucocorticoids in pregnancy, parturition, lactation, and nurturing in melanocortin receptor 2-deficient mice.

Maternal glucocorticoids are critical for fetal development, but overexpression can be deleterious. Previously we established a mouse line deficient in melanocortin receptor 2 (MC2R). MC2R(-/-) mice have undetectable levels of corticosterone despite high levels of ACTH and defects resembling those in patients with familial glucocorticoid deficiency. Here we analyzed the role of glucocorticoids in pregnancy, parturition, lactation, and nurturing in MC2R(-/-) mice. MC2R(-/-) mice were fertile and produced normal litters when crossed with MC2R(+/+) mice. However, MC2R(-/-) females crossed with MC2R(-/-) males had no live births, and approximately 20% of the embryos at d 18.5 of pregnancy were of normal body size but were dead when born. MC2R(-/-) pregnant females crossed with MC2R(+/+) males had detectable serum corticosterone levels, suggesting the transplacental passage of corticosterone from fetus to mother. MC2R(+/-) pups delivered from MC2R(-/-) females crossed with MC2R(+/+) males mice thrived poorly with MC2R(-/-) mothers but grew to adulthood when transferred to foster mothers after birth, suggesting that MC2R(-/-) females are poor mothers or cannot nurse. MC2R(-/-) females had normal alveoli, but penetration of mammary epithelium into fat pads and expression of milk proteins were reduced. Myoepithelial cells, which force milk out of the alveoli, were fully developed and differentiated. Pup retrieval behavior was normal in MC2R(-/-) mice. Exogenous corticosterone rescued expression of milk proteins in MC2R(-/-) mothers, and the pups of treated mothers grew to adulthood. Our results reveal the importance of glucocorticoids for fetal survival late in pregnancy, mammary gland development, and milk protein gene expression.

Functional hypothalamic amenorrhea due to increased CRH tone in melanocortin receptor 2-deficient mice.

Exposure to chronic stressors results in dysregulation of the hypothalamic-pituitary-adrenal axis and a disruption in reproduction. CRH, the principal regulator of the hypothalamic-pituitary-adrenal axis induces the secretion of ACTH from the pituitary, which stimulates adrenal steroidogenesis via the specific cell-surface melanocortin 2 receptor (MC2R). Previously, we demonstrated that MC2R(-/-) mice had undetectable levels of corticosterone despite high ACTH levels. Here, we evaluated the reproductive functions of female MC2R(-/-) mice and analyzed the mechanism of the disrupted cyclicity of these mice. The expression of CRH in the paraventricular nucleus was significantly increased in MC2R(-/-) mice under nonstressed conditions. Although MC2R(-/-) females were fertile, they showed a prolonged estrous cycle. After hormonal stimulation, MC2R(-/-) females produced nearly-normal numbers of eggs, but slightly less than MC2R(+/-) females, and showed near-normal ovarian histology. During diestrus, the number of GnRH-positive cells in the medial preoptic area was significantly reduced in MC2R(-/-) females. CRH type 1 receptor antagonist restored estrous cyclicity in MC2R(-/-) females. Kisspeptin-positive areas in the arcuate nucleus were comparable, whereas kisspeptin-positive areas in the anteroventral periventricular nucleus in MC2R(-/-) females were significantly reduced compared with MC2R(+/-) females, suggesting that arcuate nucleus kisspeptin is not involved, but anteroventral periventricular nucleus kisspeptin may be involved, in the maintenance of estrous cyclicity. Our findings show that high levels of hypothalamic CRH disturb estrous cyclicity in the female animals and that the MC2R(-/-) female is a unique animal model of functional hypothalamic amenorrhea.

Non-neuronal regulation and repertoire of cholinergic receptors in organs.

Many studies on the cholinergic pathway have indicated that cholinergic receptors, which are widely expressed in various cells, play an important role in all body organs. In this review, we present the concept that cholinergic responses are regulated through a neuronal or non-neuronal mechanism. The neuronal mechanism is a system in which acetylcholine binds to cholinergic receptors on target cells through the nerves. In the non-neuronal mechanism, acetylcholine, produced by neighboring cells in an autocrine/paracrine manner, binds to cholinergic receptors on target cells. Both mechanisms subsequently lead to physiological and pathophysiological responses. We also investigated the subunits/subtypes of cholinergic receptors on target cells, physiological and pathophysiological responses of the organs via cholinergic receptors, and extracellular factors that alter the subtypes/subunits of cholinergic receptors. Collectively, this concept will elucidate how cholinergic responses occur and will help us conduct further experiments to develop new therapeutic agents.