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ATP, used in cells as an energy currency, also acts as an extracellular signaling molecule. Studies of purinergic receptor subtypes have revealed that purinergic chemical transmission plays important roles in various cell types. The vesicular nucleotide transporter (VNUT), the ninth transporter in the SLC17 organic anion transporter family, is essential for vesicular ATP storage and its subsequent release. The VNUT is localized on the membrane of secretory vesicles and actively transports ATP into vesicles using an electrochemical gradient of protons supplied by vacuolar proton ATPase (V-ATPase) as a driving force. ATP acts as a damage-associated molecular pattern (DAMPs), contributing to the persistence of chronic inflammation. Chronic inflammation induces systemic insulin resistance, which is the underlying pathology of type 2 diabetes and non-alcoholic fatty liver disease (NAFLD), ranging from simple steatosis to non-alcoholic steatohepatitis (NASH). We previously demonstrated that ATP transported in insulin granules via the VNUT negatively regulates insulin secretion. We also found that hepatocytes release ATP in a VNUT-dependent manner, which elicits hepatic insulin resistance and inflammation. VNUT-knockout mice exhibited improved glucose tolerance and were resistant to the development of high fat diet-induced NAFLD. In this article, we summarize recent advances in our understanding of the mechanism of the VNUT, the development of inhibitors, and its pathological involvement in type 2 diabetes and NAFLD. The pharmacological inhibition of the VNUT may represent a potential therapeutic approach for the treatment of metabolic diseases.

Mitochondria are highly dynamic organelles continuously undergoing fission and fusion, referred as mitochondrial dynamics (MtD), to adapt to nutritional demand. Evidence suggested that impaired MtD leads to metabolic abnormalities such as nonalcoholic steatohepatitis (NASH)-like phenotypes, which are characterized by hepatic steatosis, inflammation and fibrosis. However, how MtD is involved in the development of NASH is poorly understood. In this study, we found mitochondrial fission factor (Mff), a critical mediator of mitochondrial fission, is upregulated in the liver from mice fed high-fat diet (HFD). To explore the role of Mff in the development of NASH, we generated liver-specific deletion of Mff (MffLiKO) mice. Histological analysis revealed that mitochondria are enlarged in the liver from MffLiKO mice fed normal chow diet (NCD), which is followed by hepatocyte apoptosis, slightly increased infiltration of inflammatory macrophages, and fibrosis. However, no lipid accumulation was observed in the liver from MffLiKO mice fed NCD. That is, only the impairment of Mff-regulated mitochondrial fission did not develop NASH-like phenotypes. On the other hand, the liver of MffLiKO mice fed HFD developed NASH-like phenotype with severer steatosis. Expression of mRNAs related to endoplasmic reticulum (ER) stress was markedly upregulated in the liver from MffLiKO mice. In addition, expression of genes related to hepatic triglyceride secretion was downregulated, with reduced hepatic triglyceride secretion in MffLiKO mice in vivo and in primary cultures of Mff-deficient hepatocytes in vitro. Interestingly, thapsigargin-induced ER stress suppressed triglyceride secretion in primary cultured hepatocytes in vitro. This study demonstrates that MffLiKO develop NASH-like-phenotypes in response to HFD and suggests that impaired MtD induces ER stress and thus reduces hepatic triglyceride secretion, which may play a role in the pathogenesis of NASH. Disclosure Y. Takeichi: None. T. Miyazawa: None. S. Sakamoto: None. R. Sakamoto: None. M. Nomura: None. Y. Ogawa: Research Support; Self; Mitsubishi Tanabe Pharma Corporation. Speaker’s Bureau; Self; Daiichi Sankyo, Mitsubishi Tanabe Pharma Corporation.

Vesicular nucleotide transporter (VNUT) is the last identified member of the SLC17 organic anion transporter family, which plays a central role in vesicular storage in ATP-secreting cells. The discovery of VNUT demonstrated that, despite having been neglected for a long time, vesicular ATP release represents a major pathway for purinergic chemical transmission, which had been mainly attributed to ATP permeation channels. This article summarizes recent advances in our understanding of the mechanism of VNUT and its physiopathological roles as well as the development of inhibitors. Regulating the activity and/or the expression of VNUT represents a new and promising therapeutic strategy for the treatment of multiple diseases.

Patients with type I diabetes, which is caused by the destruction of pancreatic islets, now require regular therapeutic injections of insulin. The use of transgene therapy represents an alternate and potent strategy for the treatment of type I diabetes. However, only a limited number of studies regarding in vivo gene delivery targeting the pancreas and islets have been reported. Here, we report on the possibility of in vivo transgene expression in the pancreas by the intraductal injection of naked plasmid DNA (pDNA). Gene expression activities were detected in the pancreas of mice after the injection of naked pDNA encoding luciferase into the common bile duct. We then investigated the effects of injection dose, volume, and speed on gene delivery and determined the optimal conditions for the delivery of pDNA to the pancreas. Exogenous luciferase mRNA was detected in the pancreatic islets by reverse transcription PCR analysis. Moreover, no injury was detected in the liver, the common bile duct, or the pancreas over time after the injection. These findings indicate that the intraductal injection of naked pDNA promises to be a useful technique for in vivo gene delivery targeted to pancreatic tissue and islets.

Lateral lymph node dissection (LLND) is performed for advanced rectal cancers in Japan; however, it can cause sexual and urinary dysfunction. The incidence of lateral LN metastasis is estimated at 7–13.9%; therefore, excessive rectal surgery with LLND should be avoided, especially for prophylactic purposes. To identify the patients who require LLND, we examined metastases in perirectal LNs by using a one-step nucleic acid amplification (OSNA) assay to predict lateral LN metastases. Twenty-five patients who underwent surgery with bilateral LN dissection due to T3–T4 rectal cancers were prospectively included in this study. Twenty-two patients (88.0%) received preoperative chemotherapy. Among 1052 LNs from 25 patients (median 40 per case), 135 perirectal LNs (median 6 per patient) were divided into three pieces and analyzed by OSNA, reverse transcriptase-polymerase chain reaction for carcinoembryonic antigen mRNA, and pathological examination after surgery. These results were compared with the pathological diagnosis of lateral LNs. Lateral LN metastases were present in 4 of 25 patients (16.0%). All of these patients were positive by OSNA for perirectal LN metastases. The OSNA assay had a sensitivity of 100%, specificity of 86%, positive predictive value of 57%, and negative predictive value (NPV) of 100% for predicting lateral LN metastases. The findings from this prospective study suggest that the OSNA assay of perirectal LNs may be useful for determining when LLND is necessary because of its high NPV, even in patients treated with preoperative chemotherapy.

Glutamate-mediated excitotoxicity induces neuronal death by altering various intracellular signaling pathways and is implicated as a common pathogenic pathway in many neurodegenerative diseases. In the case of motor neuron disease, there is significant evidence to suggest that the overactivation of AMPA receptors due to deficiencies in the expression and function of glial glutamate transporters GLT1 and GLAST plays an important role in the mechanisms of neuronal death. However, a causal role for glial glutamate transporter dysfunction in motor neuron death remains unknown. Here, we developed a new animal model of excitotoxicity by conditionally deleting astroglial glutamate transporters GLT1 and GLAST in the spinal cords of mice (GLAST(+/−)/GLT1-cKO). GLAST(+/−)/GLT1-cKO mice (both sexes) exhibited nuclear irregularity and calpain-mediated degradation of nuclear pore complexes (NPCs), which are responsible for nucleocytoplasmic transport. These abnormalities were associated with progressive motor neuron loss, severe paralysis, and shortened lifespan. The nuclear export inhibitor KPT-350 slowed but did not prevent motor neuron death, whereas long-term treatment of the AMPA receptor antagonist perampanel and the calpain inhibitor SNJ-1945 had more persistent beneficial effects. Thus, NPC degradation contributes to AMPA receptor-mediated excitotoxic motor neuronal death, and preventing NPC degradation has robust protective effects. Normalization of NPC function could be a novel therapeutic strategy for neurodegenerative disorders in which AMPA receptor-mediated excitotoxicity is a contributory factor. SIGNIFICANCE STATEMENT Despite glial glutamate transporter dysfunction leading to excitotoxicity has been documented in many neurological diseases, it remains unclear whether its dysfunction is a primary cause or secondary outcome of neuronal death at disease state. Here we show the combined loss of glial glutamate transporters GLT1 and GLAST in spinal cord caused motor neuronal death and hindlimb paralysis. Further, our novel mutant exhibits the nuclear irregularities and calpain-mediated progressive nuclear pore complex degradation. Our study reveals that glial glutamate transporter dysfunction is sufficient to cause motor neuronal death in vivo.

Previously, we have identified 16 senescence-associated genes by a subtractive proteomic analysis using presenescent and senescent human fibroblast cells, TIG-1. The aim of this study was to clarify the role of SMARCD1, one of the identified genes, also known as BAF60a, in hepatic senescence. SMARCD1 is a member of the SWI/SNF chromatin remodeling complex family, and regulates the transcription of target genes through the alterations of chromatin structure. We demonstrated that the reduced expression of SMARCD1 triggers cellular senescence and induces the accumulation of lipids, suggesting that SMARCD1 acts as a mediator in these processes. Furthermore, palmitic acid treatment and high-fat diet led to a significant reduction of SMARCD1 expression, and consequently induced cellular senescence and lipid accumulation in HepG2 cells and mouse liver, respectively. The results obtained here suggest that dietary nutrient-associated impaired expression of SMARCD1 triggers cellular senescence and lipid accumulation, indicating a potential application of SMARCD1 in the prevention of lifestyle-related diseases., Cellular senescence: SMARCD1, a key molecule in lipid accumulation A team led by Yoshinori Katakura at Kyushu University tried to clarify the molecular mechanisms of pathogenesis of fatty liver, focused on SMARCD1, one of the identified senescence-associated genes, and revealed its pivotal roles in cellular senescence induction and lipid accumulation in hepatocytes. SMARCD1 is a member of the SWI/SNF chromatin remodeling complex family and known to regulate the transcription of target genes through the alteration of chromatin structure. The results obtained here suggest that dietary imbalance such as high-fat diet impaires expression of SMARCD1, which triggers cellular senescence and lipid accumulation in hepatocytes, indicating a potential role of SMARCD1 in the prevention of lifestyle-related diseases.

Vesicular storage of ATP is one of the processes initiating purinergic chemical transmission. Although an active transport mechanism was postulated to be involved in the processes, a transporter(s) responsible for the vesicular storage of ATP remained unidentified for some time. In 2008, SLC17A9, the last identified member of the solute carrier 17 type I inorganic phosphate transporter family, was found to encode the vesicular nucleotide transporter (VNUT) that is responsible for the vesicular storage of ATP. VNUT transports various nucleotides in a membrane potential-dependent fashion and is expressed in the various ATP-secreting cells. Mice with knockout of the VNUT gene lose vesicular storage and release of ATP from neurons and neuroendocrine cells, resulting in blockage of the initiation of purinergic chemical transmission. Thus, VNUT plays an essential role in the vesicular storage and release of ATP. The VNUT knockout mice exhibit resistance for neuropathic pain and a therapeutic effect against diabetes by way of increased insulin sensitivity. Thus, VNUT inhibitors and suppression of VNUT gene expression may be used for therapeutic purposes through suppression of purinergic chemical transmission. This review summarizes the studies to date on VNUT and discusses what we have learned about the relevance of vesicular ATP release as a potential drug target.

The TGF-β/Smad and the PI3K/AKT signaling pathways are important regulators of proliferation and apoptosis, and their alterations lead to cancer development. TGF-β acts as a tumor suppressor in premalignant cells, but it is a tumor promoter for cancerous cells. Such dichotomous actions are dictated by different cellular contexts. Here, we have unveiled a PTEN-Smad3 regulatory loop that provides a new insight in the complex cross talk between TGF-β/Smad and PI3K/AKT signaling pathways. We demonstrate that TGF-β triggers apoptosis of wild-type polarized endometrial epithelial cells by a Smad3-dependent activation of PTEN transcription, which results in the inhibition of PI3K/AKT signaling pathway. We show that specific Smad3 knockdown or knockout reduces basal and TGF-β-induced PTEN expression in endometrial cells, resulting in a blockade of TGF-β-induced apoptosis and an enhancement of cell proliferation. Likewise Smad3 deletion, PTEN knockout prevents TGF-β-induced apoptosis and increases cell proliferation by increasing PI3K/AKT/mTOR signaling. In summary, our results demonstrate that Smad3-PTEN signaling axis determine cellular responses to TGF-β. Supported by grants SAF2016-80157-R from Ministerio de Economía y Competitividad, PI13/00263 and PI13/01701 from Fondo de Investigaciones Sanitarias del Instituto de Salud Carlos III cofinanciado por Fondo Europeo de Desarrollo Regional (FEDER) (“Una manera de hacer Europa”), Red Temática de investigación en Cáncer RD12/0036/0013 and Red de Oncología (CIBERONC). Grups consolidats de la Generalitat de Catalunya (2009SGR794), Fundació La Marató de TV3, Grupos estables AECC, Catalunya contra el cáncer and programa de intensificación de la investigación, Instituto Carlos III.

Mitochondria undergo morphological changes through fusion and fission for their quality control, which are vital for neuronal function. In this study, we examined three-dimensional morphologies of mitochondria in motor neurons under normal, nerve injured, and nerve injured plus fission-impaired conditions using the focused ion beam/scanning electron microscopy (FIB/SEM), because the FIB/SEM technology is a powerful tool to demonstrate both 3D images of whole organelle and the intra-organellar structure simultaneously. Crossing of dynamin-related protein 1 (Drp1) gene-floxed mice with neuronal injury-specific Cre driver mice, Atf3:BAC Tg mice, allowed for Drp1 ablation specifically in injured neurons. FIB/SEM analysis demonstrated that somatic mitochondrial morphologies in motor neurons were not altered before or after nerve injury. However, the fission impairment resulted in prominent somatic mitochondrial enlargement, which initially induced complex morphologies with round regions and long tubular processes, subsequently causing a decrease in the number of processes and further enlargement of the round regions, which eventually resulted in big spheroidal mitochondria without processes. The abnormal mitochondria exhibited several degradative morphologies: local or total cristae collapse, vacuolization, and mitophagy. These suggest that mitochondrial fission is crucial for maintaining mitochondrial integrity in injured motor neurons, and multiple forms of mitochondria degradation may accelerate neuronal degradation.

Background Increased urinary free cortisol in response to the oral administration of dexamethasone is a paradoxical reaction mainly reported in patients with primary pigmented nodular adrenocortical disease. Here, we describe the first case of subclinical Cushing’s syndrome represented by autonomous cortisol secretion and paradoxical response to oral dexamethasone administration, harboring an activating mutation in the α subunit of the stimulatory G protein (GNAS). Case presentation A 65-year-old woman was diagnosed with subclinical Cushing’s syndrome during an evaluation for bilateral adrenal masses. Tumors of unknown origin were found in the heart, brain, thyroid gland, colon, pancreas, and both adrenal glands. Adenocarcinoma of the sigmoid colon and systemic brown-patchy skin pigmentation were also present. Her urinary cortisol levels increased in response to oral dexamethasone, while serum dehydroepiandrosterone-sulfate was not suppressed. After right adrenalectomy, genetic analysis of the resected tumor revealed the somatic GNAS activating mutation, p.R201H. Paradoxical urinary cortisol response persisted even after unilateral adrenal resection, although serum and urinary cortisol levels were attenuated. Conclusions This patient harbored a GNAS activating mutation, and presented with a mild cortisol- and androgen-producing adrenal adenoma. Administration of oral dexamethasone paradoxically increased cortisol levels, possibly via the stimulation of the cyclic adenosine monophosphate-dependent protein kinase A signaling pathway, which is seen in patients with pigmented nodular adrenocortical disease or Carney complex. GNAS mutations may provide clues to the mechanisms of hyper-function and tumorigenesis in the adrenal cortex, especially in bilateral adrenal masses accompanied by multiple systemic tumors. Examining GNAS mutations could help physicians detect extra-adrenal malignancies, which may contribute to an improved prognosis for patients with this type of Cushing’s syndrome.

Skeletal muscle atrophy promotes muscle weakness, limiting activities of daily living. However, mechanisms underlying atrophy remain unclear. Here, we show that skeletal muscle immobilization elevates Smad2/3 protein but not mRNA levels in muscle, promoting atrophy. Furthermore, we demonstrate that myostatin, which negatively regulates muscle hypertrophy, is dispensable for denervation-induced muscle atrophy and Smad2/3 protein accumulation. Moreover, muscle-specific Smad2/3-deficient mice exhibited significant resistance to denervation-induced muscle atrophy. In addition, expression of the atrogenes Atrogin-1 and MuRF1, which underlie muscle atrophy, did not increase in muscles of Smad2/3-deficient mice following denervation. We also demonstrate that serum starvation promotes Smad2/3 protein accumulation in C2C12 myogenic cells, an in vitro muscle atrophy model, an effect inhibited by IGF1 treatment. In vivo, we observed IGF1 receptor deactivation in immobilized muscle, even in the presence of normal levels of circulating IGF1. Denervation-induced muscle atrophy was accompanied by reduced glucose intake and elevated levels of branched-chain amino acids, effects that were Smad2/3-dependent. Thus, muscle immobilization attenuates IGF1 signals at the receptor rather than the ligand level, leading to Smad2/3 protein accumulation, muscle atrophy, and accompanying metabolic changes.

Dehydroepiandrosterone (DHEA) exerts a wide variety of therapeutic effects against medical disorders, such as diabetes and obesity. However, the molecular basis of DHEA action remains to be clarified. Previously, we reported that DHEA-enhanced dual specificity protein phosphatase, designated DDSP, is one of the target molecules of DHEA. To examine the role of DDSP in DHEA signaling, we generated mice that carry a DDSP transgene in which expression is driven by the CAG promoter (DDSP-Tg). DDSP-Tg mice weighed significantly less than wild-type (WT) control mice when a high fat diet was supplied (p

Sweet taste thresholds are positively related to plasma leptin levels in normal weight humans: both show parallel diurnal variations and associations with postprandial glucose and insulin rises. Here, we tested whether this relationship also exists in overweight and obese (OW/Ob) individuals with hyperleptinemia. We tested 36 Japanese OW/Ob subjects (body mass index (BMI) > 25 kg/m2) for recognition thresholds for various taste stimuli at seven different time points from 8:00 a.m. to 10:00 p.m. using the staircase methodology, and measured plasma leptin, insulin, and blood glucose levels before each taste threshold measurement. We also used the homeostatic model assessment of insulin resistance (HOMA-IR) to evaluate insulin resistance. The results demonstrated that, unlike normal weight subjects, OW/Ob subjects showed no significant diurnal variations in the recognition thresholds for sweet stimuli but exhibited negative associations between the diurnal variations of both leptin and sweet recognition thresholds and the HOMA-IR scores. These findings suggest that in OW/Ob subjects, the basal leptin levels (~20 ng/mL) may already exceed leptin’s effective concentration for the modulation of sweet sensitivity and that this leptin resistance-based attenuation of the diurnal variations of the sweet taste recognition thresholds may also be indirectly linked to insulin resistance in OW/Ob subjects.

Mitochondria and the endoplasmic reticulum (ER) physically interact by close structural juxtaposition, via the mitochondria-associated ER membrane. Inter-organelle communication between the ER and mitochondria has been shown to regulate energy metabolism and to be central to the modulation of various key processes such as ER stress. We aimed to clarify the role of mitochondrial fission in this communication. We generated mice lacking the mitochondrial fission protein dynamin-related protein 1 (DRP1) in the liver (Drp1LiKO mice). Drp1LiKO mice showed decreased fat mass and were protected from high-fat diet (HFD)-induced obesity. Analysis of liver gene expression profiles demonstrated marked elevation of ER stress markers. In addition, we observed increased expression of the fibroblast growth factor 21 (FGF21) gene through induction of activating transcription factor 4, master regulator of the integrated stress response. Disruption of mitochondrial fission in the liver provoked ER stress, while inducing the expression of FGF21 to increase energy expenditure and protect against HFD-induced obesity.

Rationale: Both fusion and fission contribute to mitochondrial quality control. How unopposed fusion affects survival of cardiomyocytes and left ventricular function in the heart is poorly understood. Objective: We investigated the role of dynamin-related protein 1 (Drp1), a GTPase that mediates mitochondrial fission, in mediating mitochondrial autophagy, ventricular function, and stress resistance in the heart. Methods and Results: Drp1 downregulation induced mitochondrial elongation, accumulation of damaged mitochondria, and increased apoptosis in cardiomyocytes at baseline. Drp1 downregulation also suppressed autophagosome formation and autophagic flux at baseline and in response to glucose deprivation in cardiomyocytes. The lack of lysosomal translocation of mitochondrially targeted Keima indicates that Drp1 downregulation suppressed mitochondrial autophagy. Mitochondrial elongation and accumulation of damaged mitochondria were also observed in tamoxifen-inducible cardiac-specific Drp1 knockout mice. After Drp1 downregulation, cardiac-specific Drp1 knockout mice developed left ventricular dysfunction, preceded by mitochondrial dysfunction, and died within 13 weeks. Autophagic flux is significantly suppressed in cardiac-specific Drp1 knockout mice. Although left ventricular function in cardiac-specific Drp1 heterozygous knockout mice was normal at 12 weeks of age, left ventricular function decreased more severely after 48 hours of fasting, and the infarct size/area at risk after ischemia/reperfusion was significantly greater in cardiac-specific Drp1 heterozygous knockout than in control mice. Conclusions: Disruption of Drp1 induces mitochondrial elongation, inhibits mitochondrial autophagy, and causes mitochondrial dysfunction, thereby promoting cardiac dysfunction and increased susceptibility to ischemia/reperfusion.

Background Nivolumab, an anti-programmed cell death-1 monoclonal antibody, has improved the survival of patients with malignant melanoma. Despite its efficacy, nivolumab inconsistently induces thyroid dysfunction as an immune-related adverse event (irAE). This study aimed to evaluate nivolumab-induced thyroid dysfunction to determine the risks and mechanisms of thyroid irAEs. Methods After excluding 10 patients, data of 24 patients with malignant melanoma (aged 17–85 years; 54% female) were retrospectively analyzed. Results Thyroid irAEs were observed in seven patients (29%). Three patients had hypothyroidism after preceding transient thyrotoxicosis, and the other four patients had hypothyroidism without thyrotoxicosis. Levothyroxine-Na replacement was required in three patients. Antithyroid antibody (ATA) titer was elevated in one of four assessable patients. The average (±SD) time to onset of thyroid irAE was 33.6 (±21.9) weeks. The administration period of nivolumab was longer in patients with thyroid irAEs than in those without thyroid irAEs (P

SummarySuccessful chemotaxis requires not only increased motility but also sustained directionality. Here, we show that, during neutrophil chemotaxis via receptors coupled with the Gi family of heterotrimeric G proteins, directional movement is regulated by mInsc, a mammalian protein distantly related to the Drosophila polarity-organizer Inscuteable. The GDP-bound, Gβγ-free Gαi subunit accumulates at the front of chemotaxing neutrophils to recruit mInsc—complexed with the Par3-aPKC evolutionarily conserved polarity complex—via LGN/AGS3 that simultaneously binds to Gαi-GDP and mInsc. Both mInsc-deficient and aPKC-blocked neutrophils exhibit a normal motile activity but migrate in an undirected manner. mInsc deficiency prevents neutrophils from efficiently stabilizing pseudopods at the leading edge; the stability is restored by wild-type mInsc, but not by a mutant protein defective in binding to LGN/AGS3. Thus, mInsc controls directional migration via noncanonical G protein signaling, in which Gβγ-free Gαi-GDP, a product from Gαi-GTP released after receptor activation, plays a central role.

The present study describes the seasonal changes in lipid components of two brown algae, Sargassum horneri (Turner) and Cystoseira hakodatensis (Yendo), with specific reference to fucoxanthin (Fx) and n-3 (omega-3) polyunsaturated fatty acids (PUFAs). Young thalli of S. horneri were collected from different places, Nesaki and Matsushima, and cultivated in the same place. Total lipids (TL) and Fx contents of S. horneri increased from October or November and reached a maximum in January and then decreased thereafter. TL and Fx of S. horneri originated from Matsushima were always higher than those originated from Nesaki, suggesting that genetic variation might be a reason for the different TL and Fx contents found in both S. horneri seeds from different origin. Similar seasonal variation in the TL and Fx content has been more clearly observed in C. hakodatensis. Maximum TL content of S. horneri and C. hakodatensis reached to more than 14 and 15 % in winter season. These values were much higher than those found in previous studies showing TL content range from 1 to 5 %. Fatty acid composition of lipids from S. horneri and C. hakodatensis also changed seasonally. Total monounsaturated fatty acids showed the lower values from January to April, while there were no big variations in total saturated fatty acids and total n-6 PUFAs. On the other hand, total n-3 PUFAs became highest in winter. Overall, S. horneri and C. hakodatensis harvested from winter to spring will be used as good n-3 PUFA source with high levels of TL and Fx.

The bladder urothelium is more than just a barrier. When the bladder is distended, the urothelium functions as a sensor to initiate the voiding reflex, during which it releases ATP via multiple mechanisms. However, the mechanisms underlying this ATP release in response to the various stretch stimuli caused by bladder filling remain largely unknown. Therefore, the aim of this study was to elucidate these mechanisms. By comparing vesicular nucleotide transporter (VNUT)-deficient and wild-type male mice, we showed that ATP has a crucial role in urine storage through exocytosis via a VNUT-dependent mechanism. VNUT was abundantly expressed in the bladder urothelium and when the urothelium was weakly stimulated (i.e. in the early filling stages), it released ATP by exocytosis. VNUT-deficient mice showed reduced bladder compliance from the early storage phase and displayed frequent urination in inappropriate places without a change in voiding function. We conclude that urothelial, VNUT-dependent ATP exocytosis is involved in urine storage mechanisms that promote the relaxation of the bladder during the early stages of filling.

Successful recovery from neuronal damage requires a huge energy supply, which is provided by mitochondria. However, the physiological relevance of mitochondrial dynamics in damaged neurons in vivo is poorly understood. To address this issue, we established unique bacterial artificial chromosome transgenic (BAC Tg) mice, which develop and function normally, but in which neuronal injury induces labelling of mitochondria with green fluorescent protein (GFP) and expression of cre recombinase. GFP-labelled mitochondria in BAC Tg mice appear shorter in regenerating motor axons soon after nerve injury compared with mitochondria in non-injured axons, suggesting the importance of increased mitochondrial fission during the early phase of nerve regeneration. Crossing the BAC Tg mice with mice carrying a floxed dynamin-related protein 1 gene (Drp1), which is necessary for mitochondrial fission, ablates mitochondrial fission specifically in injured neurons. Injury-induced Drp1-deficient motor neurons show elongated or abnormally gigantic mitochondria, which have impaired membrane potential and axonal transport velocity during the early phase after injury, and eventually promote neuronal death. Our in vivo data suggest that acute and prominent mitochondrial fission during the early stage after nerve injury is an adaptive response and is involved in the maintenance of mitochondrial and neuronal integrity to prevent neurodegeneration.

A delay in liver regeneration after partial hepatectomy (PHx) leads to acute liver injury, and such delays are frequently observed in aged patients. BubR1 (budding uninhibited by benzimidazole-related 1) controls chromosome mitotic segregation through the spindle assembly checkpoint, and BubR1 down-regulation promotes aging-associated phenotypes. In this study we investigated the effects of BubR1 insufficiency on liver regeneration in mice. Low-BubR1-expressing mutant (BubR1L/L) mice had a delayed recovery of the liver weight-to-body weight ratio and increased liver deviation enzyme levels after PHx. Microscopic observation of BubR1L/L mouse liver showed an increased number of necrotic hepatocytes and intercalated disc anomalies, resulting in widened inter-hepatocyte and perisinusoidal spaces, smaller hepatocytes and early-stage microvilli atrophy. Up-regulation of desmocollin-1 (DSC1) was observed in wild-type, but not BubR1L/L, mice after PHx. In addition, knockdown of BubR1 expression caused down-regulation of DSC1 in a human keratinocyte cell line. BubR1 insufficiency results in the impaired liver regeneration through weakened microstructural adaptation against PHx, enhanced transient liver failure and delayed hepatocyte proliferation. Thus, our data suggest that a reduction in BubR1 levels causes failure of liver regeneration through the DSC1 abnormality.

An improved multi-step gradient reverse phase high performance liquid chromatography (HPLC) method to simultaneously separate major carotenoids from natural as well as food samples was developed. Quantitative profiling of carotenoid compounds was carried out on three edible brown seaweeds (Sargassum horneri, Cystoseira hakodatensis, and Undaria pinnatifida) and three red seaweeds (Gracilaria vermiculophylla, Grateloupia asiatica, and Grateloupia livida). Fucoxanthin (Fx) was detected in all the brown seaweeds with quantities (mg g−1 dry weight [dwt]) ranging from 1.3 ± 0.3 in C. hakodatensis to 2.4 ± 0.1 in S. horneri. U. pinnatifida, commonly known as wakame, had a Fx content of 2.3 ± 0.1 mg g−1 dwt. In the case of red seaweeds, zeaxanthin (Zx) was the major carotenoid, and G. vermiculophylla had the highest Zx content (80.2 μg g−1 dwt) among the red seaweeds apart from small amounts of Fx (9.1 μg g−1 dwt). Similarly, the other two species of red seaweeds, G. asiatica and G. livida, contained (μg g−1 dwt...

p32 is an evolutionarily conserved and ubiquitously expressed multifunctional protein. Although p32 exists at diverse intra and extracellular sites, it is predominantly localized to the mitochondrial matrix near the nucleoid associated with mitochondrial transcription factor A. Nonetheless, its function in the matrix is poorly understood. Here, we determined p32 function via generation of p32-knockout mice. p32-deficient mice exhibited mid-gestation lethality associated with a severe developmental defect of the embryo. Primary embryonic fibroblasts isolated from p32-knockout embryos showed severe dysfunction of the mitochondrial respiratory chain, because of severely impaired mitochondrial protein synthesis. Recombinant p32 binds RNA, not DNA, and endogenous p32 interacts with all mitochondrial messenger RNA species in vivo. The RNA-binding ability of p32 is well correlated with the mitochondrial translation. Co-immunoprecipitation revealed the close association of p32 with the mitoribosome. We propose that p32 is required for functional mitoribosome formation to synthesize proteins within mitochondria.

Lipid droplets (LDs) are ubiquitous organelles storing neutral lipids, including triacylglycerol (TAG) and cholesterol ester. The properties of LDs vary greatly among tissues, and LD-binding proteins, the perilipin family in particular, play critical roles in determining such diversity. Overaccumulation of TAG in LDs of non-adipose tissues may cause lipotoxicity, leading to diseases such as diabetes and cardiomyopathy. However, the physiological significance of non-adipose LDs in a normal state is poorly understood. To address this issue, we generated and characterized mice deficient in perilipin 5 (Plin5), a member of the perilipin family particularly abundant in the heart. The mutant mice lacked detectable LDs, containing significantly less TAG in the heart. Particulate structures containing another LD-binding protein, Plin2, but negative for lipid staining, remained in mutant mice hearts. LDs were recovered by perfusing the heart with an inhibitor of lipase. Cultured cardiomyocytes from Plin5-null mice more actively oxidized fatty acid than those of wild-type mice. Production of reactive oxygen species was increased in the mutant mice hearts, leading to a greater decline in heart function with age. This was, however, reduced by the administration of N-acetylcysteine, a precursor of an antioxidant, glutathione. Thus, we conclude that Plin5 is essential for maintaining LDs at detectable sizes in the heart, by antagonizing lipase(s). LDs in turn prevent excess reactive oxygen species production by sequestering fatty acid from oxidation and hence suppress oxidative burden to the heart. Background: Perilipin family proteins are important in determining the properties of lipid droplets (LDs). Results: Perilipin 5-deficient mice lack detectable LDs, exhibit enhanced fatty acid oxidation, and suffer increased ROS production in the heart. Conclusion: Perilipin 5 protects the heart from oxidative burden by sequestering fatty acid from excessive oxidation. Significance: These findings may help to increase understanding of the functions of non-adipose LDs.

Suppression of IL-2 βproduction from T cells is an important process for the immune regulation by TGF-β. However, the mechanism by which this suppression occurs remains to be established. Here, we demonstrate that Smad2 and Smad3, two major TGF-β-downstream transcription factors, are redundantly essential for TGF-β-mediated suppression of IL-2 production in CD4(+) T cells using Smad2- and Smad3-deficient T cells. Both Smad2 and Smad3 were recruited into the proximal region of the IL-2 promoter in response to TGF-β. We then investigated the histone methylation status of the IL-2 promoter. Although both histone H3 lysine 9 (H3K9) and H3K27 trimethylation have been implicated in gene silencing, only H3K9 trimethylation was increased in the proximal region of the IL-2 promoter in a Smad2/3-dependent manner, whereas H3K27 trimethylation was not. The H3K9 methyltransferases Setdb1 and Suv39h1 bound to Smad3 and suppressed IL-2 promoter activity in collaboration with Smad3. Overexpression of Suv39h1 in 68-41 T cells strongly inhibited IL-2 production in response to T cell receptor stimulation irrespective of the presence or absence of TGF-β, whereas Setdb1 overexpression only slightly suppressed IL-2 production. Silencing of Suv39h1 by shRNA reverted the suppressive effect of TGF-β on IL-2 production. Furthermore, TGF-β induced Suv39h1 recruitment to the proximal region of the IL-2 promoter in wild type primary T cells; however, this was not observed in Smad2(-/-)Smad3(+/-) T cells. Thus, we propose that Smads recruit H3K9 methyltransferases Suv39h1 to the IL-2 promoter, thereby inducing suppressive histone methylation and inhibiting T cell receptor-mediated IL-2 transcription.

The protein known as Spred1 (Sprouty-related Ena/VASP homology-1 domain-containing protein) has been identified as a negative regulator of growth factor-induced ERK/mitogen-activated protein kinase activation. Spred1 has also been implicated as the target of microRNA-126 (miR126), a miRNA located within the Egfl7 gene, and is involved in the regulation of vessel development through its role in regulating VEGF signaling. In this study, we examined the role of miR126 and Spred1 in the hematopoietic system, as miR126 has been shown to be overexpressed in leukemic cells. miR126 levels were down-regulated during mast cell differentiation from bone marrow cells, whereas Spred1 expression was inversely up-regulated. Overexpression of miR126 suppressed Spred1 expression and enhanced ERK activity in primary bone marrow cells and MC9 mast cells, which were associated with elevated FceRI-mediated cytokine production. To confirm the effect of Spred1 reduction in vivo, we generated hematopoietic cell-specific Spred1-conditional knockout mice. These mice showed increased numbers of mast cells, and Spred1-deficient bone marrow-derived mast cells were highly activated by cross-linking of Fce-R stimulation as well as by IL-3 and SCF stimulation. These results suggest that Spred1 negatively regulates mast cell activation, which is modulated by miR126.

Smad2 is a critical mediator of TGF-β signals that are known to play an important role in a wide range of biological processes in various cell types. Its role in the development of the CNS, however, is largely unknown. Mice lacking Smad2 in the CNS (Smad2-CNS-KO) were generated by a Cre-loxP approach. These mice exhibited behavioral abnormalities in motor coordination from an early postnatal stage and mortality at approximately 3 weeks of age, suggestive of severe cerebellar dysfunction. Gross observation of Smad2-CNS-KO cerebella demonstrated aberrant foliations in lobule IX and X. Further analyses revealed increased apoptotic cell death, delayed migration and maturation of granule cells, and retardation of dendritic arborization of Purkinje cells. These findings indicate that Smad2 plays a key role in cerebellar development and motor function control.

Sonic hedgehog (SHH) signaling, acting in a combinatorial manner with androgen signaling, is essential for prostate patterning and development. Recently, elevated activation of SHH signaling has been shown to play important roles in proliferation, progression and metastasis of prostate cancer. In this report, we demonstrate for the first time, that GLI1, which has been shown to play a central role in SHH signaling in prostate cancer, can act as a co-repressor to substantially block androgen receptor (AR)-mediated transactivation, at least in part, by directly interacting with AR. Our observations suggest that the SHH-GLI pathway might be one of determinants governing the transition of prostate cancer from anandrogen-dependent to an androgen-independent state by compensating, or even superseding androgen signaling.

Taste cells are chemosensory epithelial cells that sense distinct taste quality such as umami, sweet, bitter, sour and salty. Taste cells utilize G protein-coupled receptors to detect umami, sweet and bitter taste whereas ion channels are responsible for detecting salty and sour taste. Among these taste receptors, taste receptor type 2, T1R2 (or Tas1r2), has been identified as a sole sweet taste receptor in mammals that mediates sweet signals upon dimerization with T1R3. However, because of limited availability of reliable antibodies and low expression level of G protein-coupled receptors, it is uneasy to identify the cell-types that express these receptors in non-taste tissues. In this study, we have generated a T1R2-LacZ reporter knock-in mouse to investigate tissue distribution of T1R2 at a single-cell level. We found that the LacZ gene expression in these mice was faithful to the expression of T1R2 in the taste tissue and in the gastrointestinal tract where T1R3 expression has been reported. Surprisingly, T1R2 expression was also found in the testis. Mice homozygous for T1R2 deletion lacked T1R2 protein analyzed by the antibody raised against T1R2 peptide sequences. In summary, the T1R2 knock-in mouse is a powerful tool to analyze the putative targets for sweeteners as well as to study the physiological roles of T1R2 in detecting sugars.

Although it has been well established that TGF-β plays a pivotal role in immune regulation, the roles of its downstream transcription factors, Smad2 and Smad3, have not been fully clarified. Specifically, the function of Smad2 in the immune system has not been investigated because of the embryonic lethality of Smad2-deficient mice. In this study, we generated T cell-specific Smad2 conditional knockout (KO) mice and unexpectedly found that Smad2 and Smad3 were redundantly essential for TGF-β–mediated induction of Foxp3-expressing regulatory T cells and suppression of IFN-γ production in CD4+ T cells. Consistent with these observations, Smad2/Smad3-double KO mice, but not single KO mice, developed fatal inflammatory diseases with higher IFN-γ production and reduced Foxp3 expression in CD4+ T cells at the periphery. Although it has been suggested that Foxp3 induction might underlie TGF-β–mediated immunosuppression, TGF-β still can suppress Th1 cell development in Foxp3-deficient T cells, suggesting that the Smad2/3 pathway inhibits Th1 cell development with Foxp3-independent mechanisms. We also found that Th17 cell development was reduced in Smad-deficient CD4+ T cells because of higher production of Th17-inhibotory cytokines from these T cells. However, TGF-β–mediated induction of RORγt, a master regulator of Th17 cell, was independent of both Smad2 and Smad3, suggesting that TGF-β regulates Th17 development through Smad2/3-dependent and -independent mechanisms.

Background and purpose: The effects of veratridine, an alkaloid found in Liliaceae plants, on tetrodotoxin (TTX)-sensitive voltage-gated Na+ channels were investigated in mouse vas deferens. Experimental approach: Effects of veratridine on TTX-sensitive Na+ currents (INa) in vas deferens myocytes dispersed from BALB/c mice, homozygous mice with a null allele of NaV1.6 (NaV1.6−/−) and wild-type mice (NaV1.6+/+) were studied using patch-clamp techniques. Tension measurements were also performed to compare the effects of veratridine on phasic contractions in intact tissues. Key results: In whole-cell configuration, veratridine had a concentration-dependent dual action on the peak amplitude of INa: INa was enhanced by veratridine (1–10 µM), while higher concentrations (≥30 µM) inhibited INa. Additionally, two membrane current components were evoked by veratridine, namely a sustained inward current during the duration of the depolarizing rectangular pulse and a tail current at the repolarization. Although veratridine caused little shift of the voltage dependence of the steady-state inactivation curve and the activation curve for INa, veratridine enhanced a non-inactivating component of INa. Veratridine caused no detectable contractions in vas deferens from NaV1.6−/− mice, although in tissues from NaV1.6+/+ mice, veratridine (≥3 µM) induced TTX-sensitive contractions. Similarly, no detectable inward currents were evoked by veratridine in NaV1.6−/− vas deferens myocytes, while veratridine elicited both the sustained and tail currents in cells taken from NaV1.6+/+ mice. Conclusions and implications: These results suggest that veratridine possesses a dual action on INa and that the veratridine-induced activation of contraction is induced by the activation of NaV1.6 channels.

Mitochondrial morphology is dynamically controlled by a balance between fusion and fission. The physiological importance of mitochondrial fission in vertebrates is less clearly defined than that of mitochondrial fusion. Here we show that mice lacking the mitochondrial fission GTPase Drp1 have developmental abnormalities, particularly in the forebrain, and die after embryonic day 12.5. Neural cell-specific (NS) Drp1(-/-) mice die shortly after birth as a result of brain hypoplasia with apoptosis. Primary culture of NS-Drp1(-/-) mouse forebrain showed a decreased number of neurites and defective synapse formation, thought to be due to aggregated mitochondria that failed to distribute properly within the cell processes. These defects were reflected by abnormal forebrain development and highlight the importance of Drp1-dependent mitochondrial fission within highly polarized cells such as neurons. Moreover, Drp1(-/-) murine embryonic fibroblasts and embryonic stem cells revealed that Drp1 is required for a normal rate of cytochrome c release and caspase activation during apoptosis, although mitochondrial outer membrane permeabilization, as examined by the release of Smac/Diablo and Tim8a, may occur independently of Drp1 activity.

Hypogonadism is associated with increased fat mass and dysregulation of metabolic homeostasis in men. Our previous study revealed that androgen receptor (AR)-null male mice (AR L-/Y ) develop late-onset obesity and are leptin-resistant. The present study evaluated how hypothalamic AR contributes to central leptin-signal transducer and activator of transcription 3 (STAT3) signaling. We evaluated leptin action in wild-type and AR L-/Y mice, the anatomic co-relationship between AR and leptin signaling in the hypothalamus, and the effects of AR on leptin-mediated STAT3 transactivation and nuclear translocation. AR deletion in male mice results in a weaker leptin-induced suppression of food intake and body weight drop even before the onset of overt obesity. In wildtype male but not female mice, AR was highly expressed in various hypothalamic nuclei that also expressed the longform leptin receptor (OBRB) and co-resided with OBRB directly in the arcuate neurons. In vitro, AR significantly enhanced STAT3-mediated transcription of leptin target genes including POMC and SOCS3. This effect relied on the AR N-terminal activation function-1 (AF-1) domain and was specific to AR in that none of the other sex steroid hormone receptors tested showed similar effects. AR enhanced the low concentrations of leptin-induced STAT3 nuclear translocation in vitro, and AR L-/Y mice receiving leptin had impaired STAT3 nuclear localization in the arcuate neurons. These findings indicate that AR in the hypothalamus functions as a regulator of central leptin-OBRB-STAT3 signaling and has a physiological role in energy homeostasis and metabolic regulation in male mice. (Endocrinology 149: 6028–6036, 2008)

Summary Glucocorticoids are lipid substances synthesized by the adrenal cortex that are essential for life and regulate myriad physiological processes ubiquitously in organs and tissues as mediated by intracellular glucocorticoid receptors (GRs). From the clinical standpoint, although glucocorticoids have potent anti-inflammatory and immunosuppressive effects, their adverse effects profiles and so-called glucocorticoid resistance are barriers to their widespread use. GRβ, a splice variant isoform of GRα, the predominant isoform of the receptor, and co-activators and co-repressors are believed to be important for GR-mediated actions. The mechanism of action of GRα and its co-activators and co-repressors and the mechanism of resistance to glucocorticoid treatment were investigated by confocal microscopic imaging of GRα and GRβ and by assessing protein–protein interaction of GRα and nuclear factor-κB and of GRα and activator protein-1 (AP-1). The possibility of new drug development of selective GR modulators, which reduce GR-related adverse effects such as steroid-induced osteoporosis, steroid-induced diabetes mellitus, and infection yet confer beneficial anti-inflammatory effects and immunosuppressive action is discussed.

Signaling through activin type IIB receptor (ActRIIB) has been shown to regulate the axial formation and the development of foregut-derived organs such as the pancreas in mice. Here, we provide genetic evidence that ActRIIB and Smad2 genes cooperatively regulated asymmetrical patterning of the thoracic organs and pancreas development in mice. The loss of one allele of Smad2 on ActRIIB−/− background resulted in the increased severity of ActRIIB−/− phenotypes, including right pulmonary isomerism and complex cardiac malformations, and resulted in 100% frequency of death soon after birth. Of interest, 14% of compound heterozygous ActRIIB+/−Smad2+/− mice exhibited the ActRIIB−/− phenotypes and died soon after birth. In the pancreas, hypoplastic islets were found not only in ActRIIB−/− but also in Smad2+/− mice. A more severe phenotype was also found in ActRIIB+/−Smad2+/− mice. As well, these mutant mice exhibited impaired glucose tolerance in a gene dosage-sensitive manner. This genetic evidence strongly suggested that ActRIIB and Smad2 function in the same signaling pathway to regulate axial patterning and pancreas islet formation by means of a threshold mechanism. Developmental Dynamics 236:2865–2874, 2007. © 2007 Wiley-Liss, Inc.

Type 2 diabetes is closely associated with our daily diets and has become a global health problem with increasing number of patients. Maintaining energy homeostasis is essentially required for the treatment of diabetes. Energy metabolism starts with taking in a meal. Nutrients including amino acids, fatty acids and glucose in the digest have been shown to act on the neuroendocrine cells in the gastrointestinal (GI) tract, and thereby play important roles in energy homeostasis. Therefore, the GI tract is now recognized as a sensor system for nutrient signals. Taste receptor type 1 member 2 (T1R2) is known to function as a co-receptor with T1R3 to detect sweet chemicals in the taste buds. It has been proposed that the T1R2/T1R3 receptor complex acts as sweet sensor in the intestine, and plays a pivotal role in sensing sugars and maintaining glucose homeostasis through incretin secretion. To clarify the physiological roles of T1R2 in glucose homeostasis, T1r2-lacZ knock-in/knock-out mice were generated. We found lacZ gene expression in the GI tract where T1r3 expression has been reported. Interestingly, the T1r2-lacZ knock-in mice showed impaired glucose tolerance on oral glucose challenge but not on intraperitoneal injection. However, the fasting glucose level in T1r2-lacZ knock-in mice was comparable to that in wild type mice. These results suggest an important role of the sweet taste receptor system in the intestine when stimulated by glucose. Therefore, the roles of T1R2 will be presented and the mechanism for metabolic homeostasis will be discussed.

Mitochondria are dynamic organelles, and their fusion and fission regulate cellular signaling, development, and mitochondrial homeostasis, including mitochondrial DNA (mtDNA) distribution. Cardiac myocytes have a specialized cytoplasmic structure where large mitochondria are aligned into tightly packed myofibril bundles; however, recent studies have revealed that mitochondrial dynamics also plays an important role in the formation and maintenance of cardiomyocytes. Here, we precisely analyzed the role of mitochondrial fission in vivo. The mitochondrial fission GTPase, Drp1, is highly expressed in the developing neonatal heart, and muscle-specific Drp1 knockout (Drp1-KO) mice showed neonatal lethality due to dilated cardiomyopathy. The Drp1 ablation in heart and primary cultured cardiomyocytes resulted in severe mtDNA nucleoid clustering and led to mosaic deficiency of mitochondrial respiration. The functional and structural alteration of mitochondria also led to immature myofibril assembly and defective cardiomyocyte hypertrophy. Thus, the dynamics of mtDNA nucleoids regulated by mitochondrial fission is required for neonatal cardiomyocyte development by promoting homogeneous distribution of active mitochondria throughout the cardiomyocytes.

Introduction: The mitochondrial dynamic processes of fission and fusion influence and integrate with multiple physiologic and pathophysiologic processes. Mitochondrial dynamics dysregulation has been implicated in atherosclerosis, but little is known about the role of myeloid cell specific mitochondrial dynamics in the progression of atherosclerosis. In macrophage-enriched murine atherosclerosis lesion areas, we have found that levels of mitochondrial fission protein DRP1 down-regulated as the lesion progresses. In contrast, the mitochondrial fusion protein MFN2 is up-regulated. Further, mitochondria in lesional macrophages show hyperfusion morphology as the lesion develops. These suggest that mitochondria in macrophages undergo hyperfusion during the lesion progression. Hypothesis: We hypothesize that mitochondrial hyperfusion plays a significant role in atherosclerosis. Methods: We used a model Drp1fl/fl LysmCre+/-Ldlr-/-mice who have hyperfused mitochondria in Mϕs to test the functional significance of mitochondrial hyperfusion in atherosclerosis. Results: We have found that inhibition of Mϕ mitochondrial fission leads to a striking increase of necrotic core area and the accumulation of apoptotic cells, which are likely due to the defective phagocytic clearance of apoptotic cells (efferocytosis) in the advanced stage of atherosclerosis in vivo. This is further verified by another in vivo efferocytosis assay: Drp1fl/fl LysmCre+/-mice are defective of clearing apoptotic thymocytes in vivo. Mechanistically, the continued uptake of apoptotic cellsis impaired in Mϕs with hyperfused mitochondria. This is because of the lower level of uncoupling protein 2 (UCP2), the mitochondrial inner membrane protein that prevents the sustained elevation of inner membrane potential (Δψ). Chemical uncoupler FCCP or restoration of UCP2 can correct the efferocytosis deficiency in DRP1 knockout Mϕs. Conclusions: Macrophage mitochondrial fission is essential for continued clearance of apoptotic cells and plays a protective role in advanced atherosclerosis. This study indicates that mitochondrial fusion/fission could be a novel therapeutic target to prevent lesion necrosis and stabilize the advanced plaques in humans.

Androgen signaling plays key roles in the development and progression of prostate cancer, and numerous ongoing studies focus on the regulation of androgen receptor (AR) transactivity to develop novel therapies for the treatment of androgen-independent prostate cancer. FoxH1, a member of the Forkhead-box (FOX) gene family of transcription factors, takes part in mediating transforming growth factor-β/activin signaling through its interaction with the Smad2·Smad4 complex. Using a series of experiments, we found that FoxH1 repressed both ligand-dependent and -independent transactivation of the AR on androgen-induced promoters. This action of FoxH1 was independent of its transactivation capacity and activin A but relieved by Smad2·Smad4. In addition, the repression of the AR by FoxH1 did not require deacetylase activity. A protein-protein interaction was identified between the AR and FoxH1 independently of dihydrotestosterone. Furthermore, a confocal microscopic analysis of LNCaP cells revealed that the interaction between the AR and FoxH1 occurred in the nucleus and that FoxH1 specifically blocked the foci formation of dihydrotestosterone-activated AR, which has been shown to be correlated with the AR transactivation potential. Taken together, our results indicate that FoxH1 functions as a new corepressor of the AR. Our observations not only strengthen the role of FoxH1 in AR-mediated transactivation but also suggest that therapeutic interventions based on AR-coregulator interactions could be designed to block both androgen-dependent and -independent growth of prostate cancer.

Bone marrow stem cells develop into haematopoietic and mesenchymal lineages, but have not been known to participate in steroidogenic cell production. Steroidogenic factor 1 (SF-1), also designated adrenal 4 binding protein (Ad4BP), is an essential orphan nuclear receptor for steroidogenesis as well as for adrenal and gonadal gland development. In the present study, we revealed that the adenovirus-mediated forced expression of SF-1 can transform cultured primary long-term cultured bone marrow cells into steroidogenic cells, showing the de novo synthesis of multiple steroid hormones in response to adrenocorticotropic hormone (ACTH). This finding may provide an initial step in innovative autograft cell transfer therapy for steroid hormone deficiencies.

Endocrine disruptor chemicals are known to cause a range of abnormalities in sexual differentiation and reproduction. One mechanism underlying such effects may be via alteration of aromatase activity, which is responsible for estrogen production. A good screening system for identifying endocrine disruptors has long been desired. We have recently established a human ovarian granulosa-like tumor cell line, KGN, which possesses a relatively high level of aromatase expression and is considered a useful mammalian model for investigating the in vitro effects of various chemicals on aromatase activity. In this study we screened 55 different candidate chemicals for endocrine disruptors by assaying aromatase activity. Only benomyl, known as both a benzimidazole fungicide and a microtubule-interfering agent, was found to induce aromatase activity in association with increased levels of aromatase mRNA in KGN cells. The effect of benomyl was presumed to be mediated by its metabolite carbendazim, because it produced an effect equivalent to that of benomyl. The mechanism underlying the benomyl-induced increase in aromatase activity appears independent of the cAMP-protein kinase A pathway. Treatment with taxol, another class of microtubule-interfering agents, also caused induction of aromatase in KGN cells. Both benomyl and taxol changed KGN cell morphology, including the development of cell roundness and a disorganized network of microtubules. These results indicate that benomyl is a potential endocrine disruptor that provides a novel estrogenicity and operates through a microtubule-interfering mechanism.

The mechanism through which protein kinase A (PKA) potentiates the transactivation ability of adrenal 4 binding protein/steroidogenic factor 1 (Ad4BP/SF-1) is currently unclear. In the present study, we investigated the mechanism by applying laser confocal microscopy and fluorescence recovery after photobleaching technique. In KGN cells, forskolin (a PKA stimulator) could reorganize wild-type Ad4BP/SF-1, but not mutant Ad4BP/SF-1 (G35E), from a diffuse distribution pattern to foci formation in the nucleus. The subcellular distributions of GCN5 (general control nonderepressed) and TRRAP (transformation/transcription domain-associated protein), both of which were recently proved to be working in the same complex as the third class of nuclear receptor coactivators, were unexpectedly diffuse inside and outside the nucleus, respectively, when they were separately transfected. However TRRAP was translocated into the nucleus in the presence of GCN5, and together with GCN5 colocalized with Ad4BP/SF-1 in the same foci when PKA was activated. A luciferase assay also indicated that these two cofactors enhanced Ad4BP/SF-1 transactivation.Dosage-sensitive sex reversal (DAX-1) interacts with and thus inhibits Ad4BP/SF-1 transactivation. The coexistence of the two proteins dramatically altered their respective subnuclear distributions. They colocalized extensively, suggestive of binding, and Ad4BP/SF-1 was sharply immobilized when DAX-1 was coexpressed, whereas PKA could maintain mobility, as evidenced by Fluorescence Recovery After Photobleaching showing that Ad4BP/SF-1 mobility recovered after forskolin treatment.Therefore, the PKA signal pathway may modify the interaction between Ad4BP/SF-1 and its activators and repressor (GCN5 and TRRAP are integrated, whereas DAX-1 is disassociated), and thus stimulate the Ad4BP/SF-1 transactivation.

We have mainly focused on the regulatory mechanism of cytochrome P450 aromatize in bone cells. Our previous study demonstrated a strong positive correlation of serum dehydroepiandrosterone sulfate (DHEA-S) and estrone (E1) with BMD in postmenopausal women but no correlation between serum estradiol (E2) and BMD in the same group. In addition, administration of DHEA to ovariectomized rat significantly increased BMD. These in vivo findings strongly suggested that circulating adrenal androgen may be converted to estrogen in osteoblast and may contribute to BMD maintenance. Actually, in cultured human osteoblast cells, DHEA was found to convert to androstenedione by 3beta-hydroxysteroid dehydrogenase (3beta-HSD) activity and then androstenedione to estrone through the apparent aromatase activity. The aromatase activity in cultured human osteoblast cells was significantly increased by dexamethasone (DEX). Interestingly, DEX and 1alpha,25-dihydroxyvitamin D3 (VD3) synergistically enhanced aromatase activity as well as P450arom mRNA expression. A little stronger induction of aromatase activity by DEX and VD3 was observed in cultured human fibroblasts. The increase of the aromatase activity by DEX and VD3 was accompanied with the increase of luciferase activity of fibroblast cells transfected with Exon 1b-promoter-luciferase construct, but not of osteoblasts transfected with the same construct, suggesting a different regulatory mechanism of aromatase by DEX and 1alpha,25-dihydroxyvitamin D3 (VD3) between these two cells despite the same promotor usage. In human bone cells, intracrine mechanism through aromatase activity, together with a positive regulation of aromatase activity by glucocorticoid and VD3, may contribute to the local production of estrogens, thus leading to protective effect against osteoporosis especially after menopause. The effect of sex steroids (estrogen versus testosterone) in bone remodeling was also briefly reviewed based on several recent findings in this field.

Pheochromocytoma crisis is a life-threatening endocrine emergency associated with symptoms of excess release of catecholamines. It might present spontaneously or be unmasked by triggers including trauma, surgery and certain medications that provoke catecholamine release by tumors. Here we report a case of pheochromocytoma crisis associated with abscess formation in the tumor and bacteremia of Campylobacter fetus, which was successfully treated with antibiotics and a surgical resection. This case appears to be the first reported case in the literature of abscess formation by C. fetus in pheochromocytoma, leading to catecholamine crisis.

In the androgen receptor (AR), most of its transactivation activity is mediated via the activation function-1 (AF-1). By employing yeast two-hybrid assay, we isolated a cDNA sequence encoding a protein binding to AR-AF-1. This protein, named ANT-1 (AR N-terminal domain transactivating protein-1), enhanced the ligand-independent autonomous AF-1 transactivation function of AR or glucocorticoid receptor but did not enhance that of estrogen receptor alpha. In contrast, the ANT-1 did not enhance any ligand-dependent AF-2 activities. Furthermore, the ligand-independent interaction between AR-AF-1 and ANT-1 was confirmed in vivo and in vitro. The ANT-1 sequence was identical to that of a protein that binds to U5 small nuclear ribonucleoprotein particle, a human homologue of yeast splicing factor Prp6p, involved in spliceosome. ANT-1 was compartmentalized into 20-40 coarse splicing factor compartment speckles against the background of the diffuse reticular distribution. AR colocalized with ANT-1 only in the diffusely distributed area, whereas the ANT-1 speckles were spatially distinct from but surrounded by the AR compartments. The active gene transcription has been shown to couple simultaneously with pre-mRNA processing at the periphery of the splicing factor compartment. The molecular interaction between two spatially distinct subnuclear compartments mediated by ANT-1 may therefore recruit AR into the transcription-splicing-coupling machinery.

Summary Clearance of apoptotic cells (ACs) by phagocytes (efferocytosis) prevents post-apoptotic necrosis and dampens inflammation. Defective efferocytosis drives important diseases, including atherosclerosis. For efficient efferocytosis, phagocytes must be able to internalize multiple ACs. We show here that uptake of multiple ACs by macrophages requires dynamin-related protein 1 (Drp1)-mediated mitochondrial fission, which is triggered by AC uptake. When mitochondrial fission is disabled, AC-induced increase in cytosolic calcium is blunted owing to mitochondrial calcium sequestration, and calcium-dependent phagosome formation around secondarily encountered ACs is impaired. These defects can be corrected by silencing the mitochondrial calcium uniporter (MCU). Mice lacking myeloid Drp1 showed defective efferocytosis and its pathologic consequences in the thymus after dexamethasone treatment and in advanced atherosclerotic lesions in fat-fed Ldlr −/− mice. Thus, mitochondrial fission in response to AC uptake is a critical process that enables macrophages to clear multiple ACs and to avoid the pathologic consequences of defective efferocytosis in vivo .

Neuroendocrine cells store ATP in secretory granules and release it along with hormones that may trigger a variety of cellular responses in a process called purinergic chemical transmission. Although the vesicular nucleotide transporter (VNUT) has been shown to be involved in vesicular storage and release of ATP, its physiological relevance in vivo is far less well understood. In Vnut knockout (Vnut(-/-)) mice, we found that the loss of functional VNUT in adrenal chromaffin granules and insulin granules in the islets of Langerhans led to several significant effects. Vesicular ATP accumulation and depolarization-dependent ATP release were absent in the chromaffin granules of Vnut(-/-) mice. Glucose-responsive ATP release was also absent in pancreatic β-cells in Vnut(-/-) mice, while glucose-responsive insulin secretion was enhanced to a greater extent than that in wild-type tissue. Vnut(-/-) mice exhibited improved glucose tolerance and low blood glucose upon fasting due to increased insulin sensitivity. These results demonstrated an essential role of VNUT in vesicular storage and release of ATP in neuroendocrine cells in vivo and suggest that vesicular ATP and/or its degradation products act as feedback regulators in catecholamine and insulin secretion, thereby regulating blood glucose homeostasis.

In studies of gene-ablated mice, activin signaling through activin type IIB receptors (ActRIIB) and Smad2 has been shown to regulate not only pancreatic β cell mass but also insulin secretion. However, it still remains unclear whether gain of function of activin signaling is involved in the modulation of pancreatic β cell mass and insulin secretion. To identify distinct roles of activin signaling in pancreatic β cells, the Cre-loxP system was used to activate signaling through activin type IB receptor (ActRIB) in pancreatic β cells. The resultant mice (pancreatic β cell-specific ActRIB transgenic (Tg) mice; ActRIBCAβTg) exhibited a defect in glucose-stimulated insulin secretion (GSIS) and a progressive impairment of glucose tolerance. Patch-clamp techniques revealed that the activity of ATP-sensitive K(+) channels (KATP channels) was decreased in mutant β cells. These results indicate that an appropriate level of activin signaling may be required for GSIS in pancreatic β cells, and that activin signaling involves modulation of KATP channel activity.

Pancreatic beta cells secrete insulin in response to glucose levels and thus are involved in controlling blood glucose levels. A line of pancreatic beta cells "MIN6" has been used in studies related to the function of beta cells and diabetes therapy. Regulating gene expression in MIN6 cells could accelerate these studies, but an efficient method for the transfection of nucleic acids targeted to MINE cells is required. We report here on a liposome-based carrier targeted to pancreatic beta cells (Multifunctional envelope-type nano device for pancreatic beta cells, beta-MEND). We identified a lipid composition for use in preparing the beta-MEND, which permits the particles to be efficiently internalized into MIN6, as evidenced by flow cytometry analyses. Intracellular observation by confocal laser scanning microscopy showed that the beta-MEND efficiently delivered the oligo nucleic acids to the cytosol of MINE cells. Moreover, using a beta-MEND encapsulating a 2'-O-Methyl RNA complementary to a microRNA that suppresses insulin secretion, the knockdown of the targeted microRNA and an up-regulation of insulin secretion were observed in MINE. Thus, the beta-MEND holds promise as an efficient system for delivering nucleic acids targeted to MIN6 and can contribute to research and therapy aimed at diabetes. (C) 2014 Elsevier Ltd. All rights reserved.