Although mammalian target of rapamycin (mTOR) mediates a wide variety of biological functions, little information is available on the effect of mTOR on the functions of skin cells. In this study, we investigated effects of mTOR inhibition by rapamycin on ceramide synthesis in the skin of rats and human keratinocytes and its regulatory mechanisms. The phosphorylation of p70 S6 kinase, which indicates mTOR activation, was induced in the skin of rats fed a high-fat diet, but this abnormality was reversed by supplementation with rapamycin. Ceramide levels and the mRNA levels of serine palmitoyltransferase (SPT) and transforming growth factor (TGF)-β1 were suppressed in the skin of rats fed high-fat diets, but this abnormality was reversed by supplementation with rapamycin. TGF-β1-induced SPT mRNA expression was blocked by SB525334, an inhibitor of TGF-β1-induced Smad2/3 nuclear localization, in human keratinocytes. Rapamycin-induced SPT mRNA expression was blocked by an anti-TGF-β1 antibody or SB525334 in human keratinocytes. These results show that mTOR inhibition by rapamycin increases ceramide synthesis by promoting TGF-β1/Smad signaling in the skin.

Memantine is a non-competitive antagonist of the N-methyl-D-aspartate (NMDA) receptor, and is an approved drug for the treatment of moderate-to-severe Alzheimer's disease. We identified a mouse strain with a naturally occurring mutation and an ataxic phenotype that presents with severe leg cramps. To investigate the phenotypes of these mutant mice, we screened several phenotype-modulating drugs and found that memantine (10 mg/kg) disrupted the sense of balance in the mutants. Moreover, the mutant mice showed an attenuated optokinetic response (OKR) and impaired OKR learning, which was also observed in wild-type mice treated with memantine. Microsatellite analyses indicated that the Grid2 gene-deletion is responsible for these phenotypes. Patch-clamp analysis showed a relatively small change in NMDA-dependent current in cultured granule cells from Grid2 gene-deleted mice, suggesting that GRID2 is important for correct NMDA receptor function. In general, NMDA receptors are activated after the activation of non-NMDA receptors, such as AMPA receptors, and AMPA receptor dysregulation also occurs in Grid2 mutant mice. Indeed, the AMPA treatment enhanced memantine susceptibility in wild-type mice, which was indicated by balance sense and OKR impairments. The present study explores a new role for GRID2 and highlights the adverse effects of memantine in different genetic backgrounds.

Thrombin aggravates ischemic stroke and activated protein C (APC) has a neuroprotective effect. Both proteases interact with protease-activated receptor 1, which exhibits functional selectivity and leads to G-protein- and β-arrestin-mediated-biased signal transduction. We focused on the effect of β-arrestin in PAR-1-biased signaling on endothelial function after stroke or high-fat diet (HFD). Thrombin had a rapid disruptive effect on endothelial function, but APC had a slow protective effect. Paralleled by prolonged MAPK 42/44 signaling activation by APC via β-arrestin-2, a lower cleavage rate of PAR-1 for APC than thrombin was quantitatively visualized by bioluminescence video imaging. HFD-fed mice showed lower β-arrestin-2 levels and more severe ischemic injury. The expression of β-arrestin-2 in capillaries and PDGF-β secretion in HFD-fed mice were reduced in penumbra lesions. These results suggested that β-arrestin-2-MAPK-PDGF-β signaling enhanced protection of endothelial function and barrier integrity after stroke.

Mechanisms underlying the central regulation of food intake and fat accumulation are not fully understood. We found that neurosecretory protein GL (NPGL), a newly-identified neuropeptide, increased food intake and white adipose tissue (WAT) in rats. NPGL-precursor gene overexpression in the hypothalamus caused increases in food intake, WAT, body mass, and circulating insulin when fed a high calorie diet. Intracerebroventricular administration of NPGL induced de novo lipogenesis in WAT, increased insulin, and it selectively induced carbohydrate intake. Neutralizing antibody administration decreased the size of lipid droplets in WAT. Npgl mRNA expression was upregulated by fasting and low insulin levels. Additionally, NPGL-producing cells were responsive to insulin. These results point to NPGL as a novel neuronal regulator that drives food intake and fat deposition through de novo lipogenesis and acts to maintain steady-state fat level in concert with insulin. Dysregulation of NPGL may be a root cause of obesity.

Unilateral spatial neglect (USN) is one of the most common symptoms of right hemisphere damage; its classical symptom is that patients fail to respond to information on their left side. It has been postulated that disturbance of 2 separate attentional networks relates to the occurrence of USN. However, little is known about the underlying mechanism and neuronal substrates. In this study, we measured spontaneous neural activity by means of magnetoencephalography in 13 patients with brain damage and 5 control subjects. To study the relationship between functional connectivity at rest and severity of USN symptoms, we determined the imaginary coherence values relating to the inter-hemispherical ventral and dorsal attentional networks, as well as the clinical severity of USN using neuropsychological tests and behavioral rating scales. The present results showed that inter-hemispherical connectivity in the ventral attentional network, especially between the left and right angular gyri, detected in the alpha band is associated with the severity of USN symptoms. This may suggest that connectivity of inter-hemispherical homologous regions of the ventral attentional network in the alpha band could be one of the biomarkers of attentional network imbalance occurring in patients with USN.

Flavonoids, which are plant polyphenols, are now widely used in supplements and cosmetics. Here, we report that 4'-methylflavonoids are potent inducers of melanogenesis in B16F10 melanoma cells and in mice. We recently identified salt inducible kinase 2 (SIK2) as an inhibitor of melanogenesis via the suppression of the cAMP-response element binding protein (CREB)-specific coactivator 1 (TORC1). Using an in vitro kinase assay targeting SIK2, we identified fisetin as a candidate inhibitor, possibly being capable of promoting melanogenesis. However, fisetin neither inhibited the CREB-inhibitory activity of SIK2 nor promoted melanogenesis in B16F10 melanoma cells. Conversely, mono-methyl-flavonoids, such as diosmetin (4'-O-metlylluteolin), efficiently inhibited SIK2 and promoted melanogenesis in this cell line. The cAMP-CREB system is impaired in A(y)/a mice and these mice have yellow hair as a result of pheomelanogenesis, while Sik2(+/-); A(y)/a mice also have yellow hair, but activate eumelanogenesis when they are exposed to CREB stimulators. Feeding Sik2(+/-); A(y)/a mice with diets supplemented with fisetin resulted in their hair color changing to brown, and metabolite analysis suggested the presence of mono-methylfisetin in their feces. Thus, we decided to synthesize 4'-O-methylfisetin (4'MF) and found that 4'MF strongly induced melanogenesis in B16F10 melanoma cells, which was accompanied by the nuclear translocation of TORC1, and the 4'-O-methylfisetin-induced melanogenic programs were inhibited by the overexpression of dominant negative TORC1. In conclusion, compounds that modulate SIK2 cascades are helpful to regulate melanogenesis via TORC1 without affecting cAMP levels, and the combined analysis of Sik2(+/-) mice and metabolites from these mice is an effective strategy to identify beneficial compounds to regulate CREB activity in vivo.

SIR2 is an NAD(+)-dependent deacetylase [1]-[3] implicated in the regulation of lifespan in species as diverse as yeast [4], worms [5], and flies [6]. We previously reported that the level of SIRT1, the mammalian homologue of SIR2 [7], [8], is coupled to the level of mitotic activity in cells both in vitro and in vivo[9]. Cells from long-lived mice maintained SIRT1 levels of young mice in tissues that undergo continuous cell replacement by proliferating stem cells. Changes in SIRT1 protein level were not associated with changes in mRNA level, suggesting that SIRT1 could be regulated post-transcriptionally. However, other than a recent report on sumoylation [10] and identification of SIRT1 as a nuclear phospho-protein by mass spectrometry [11], post-translational modifications of this important protein have not been reported.

MicroRNA-132/212 has been supposed as a critical gene related to the blood-brain barrier (BBB) protection after stroke, but its regulation pathway including the upstream regulator and downstream targets is still unclear. Herein, we demonstrated the cAMP response element-binding protein (CREB)-regulated transcription coactivator-1 (CRTC1) to be the upstream regulator of miRNA-132/212 using CRTC1 knockout and wild-type mice. CRTC1 deletion led to the reduction of miRNA-132/212 expression in mice brain after ischemic stroke, significantly increased infarct volume, and aggravated BBB permeability with worsening neurological deficits. Furthermore, we identified that miRNA-132 repressed Claudin-1, tight junction-associated protein-1 (TJAP-1), and RNA-binding Fox-1 (RBFox-1) by directly binding to their respective 3'-untranslated regions, which alleviated the ischemic damage by enhancing neuronal survival and BBB integrity. Moreover, the co-culture of endothelial cells with CRTC1-deficient neurons aggravated the cell vulnerability to hypoxia, also supporting the idea that miRNA-132/212 cluster is regulated by CRTC1 and acts as a crucial role in the mitigation of ischemic damage. This work is a step forward for understanding the role of miRNA-132/212 in neurovascular interaction and may be helpful for potential gene therapy of ischemic stroke.

Mdmx and Mdm2 are two major suppressor factors for the tumor suppressor gene p53. In central nervous system, Mdmx suppresses the transcriptional activity of p53 and enhances the binding of Mdm2 to p53 for degradation. But Mdmx dynamics in cerebral infarction remained obscure. Here we investigated the role of Mdmx under ischemic conditions and evaluated the effects of our developed small-molecule Protein-Protein Interaction (PPI) inhibitors, K-181, on Mdmx-p53 interactions in vivo and in vitro. We found ischemic stroke decreased Mdmx expression with increased phosphorylation of Mdmx Serine 367, while Mdmx overexpression by AAV-Mdmx showed a neuroprotective effect on neurons. The PPI inhibitor, K-181 attenuated the neurological deficits by increasing Mdmx expression in post-stroke mice brain. Additionally, K-181 selectively inhibited HDAC6 activity and enhanced tubulin acetylation. Our findings clarified the dynamics of Mdmx in cerebral ischemia and provide a clue for the future pharmaceutic development of ischemic stroke.

To clarify the characteristics of cryptogenic stroke in patients with active cancer.

With increased histone deacetylase (HDAC) activity and histone hypoacetylation being implicated in neurodegeneration, HDAC inhibitors have been reported to have considerable therapeutic potential. Yet, existing inhibitors lack specificity and may show substantial adverse effect. In this study, we identified a novel HDAC1/2 isoform-specific inhibitor, K560, with protective effects against 1-methyl-4-phenylpyridinium (MPP(+))- and/or 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced neuronal death in both in vitro and in vivo Parkinson's disease model. K560 attenuated cell death induced by MPP(+) in differentiated SH-SY5Y cells through the sustained expression of an antiapoptotic protein, X-linked inhibitor of apoptosis (XIAP). Inhibition of XIAP expression by locked nucleic acid antisense oligonucleotides abolished the protective effect of K560. Inactivation of mitogen-activated protein kinase cascades, reduced p53 phosphorylation, and down-regulation of p53-upregulated modulator of apoptosis on K560 treatment were also observed. Furthermore, pre- and post-oral administration of K560 to mice prevented MPTP-induced loss of dopaminergic neurons in substantia nigra, suggesting that selective inhibition of HDAC1 and HDAC2 by K560 may pave the way to new strategies for Parkinson's disease treatment.

Hydrocellular foam dressing, modern wound dressing, induces moist wound environment and promotes wound healing: however, the regulatory mechanisms responsible for these effects are poorly understood. This study was aimed to reveal the effect of hydrocellular foam dressing on hyaluronan, which has been shown to have positive effects on wound healing, and examined its regulatory mechanisms in rat skin.

Sweet taste receptor is expressed not only in taste buds but also in nongustatory organs such as enteroendocrine cells and pancreatic beta-cells, and may play more extensive physiological roles in energy metabolism. Here we examined the expression and function of the sweet taste receptor in 3T3-L1 cells.

Nicotinamide adenine dinucleotide (NAD) is an important cofactor that regulates various biological processes, including metabolism and gene expression. As a coenzyme, NAD controls mitochondrial respiration through enzymes of the tricarboxylic acid (TCA) cycle, β-oxidation, and oxidative phosphorylation and also serves as a substrate for posttranslational protein modifications, such as deacetylation and ADP-ribosylation by sirtuins and poly(ADP-ribose) polymerase (PARP), respectively. Many studies have demonstrated that NAD levels decrease with aging and that these declines cause various aging-associated diseases. In contrast, activation of NAD metabolism prevents declines in NAD levels during aging. In particular, dietary supplementation with NAD precursors has been associated with protection against age-associated insulin resistance. However, it remains unclear which NAD synthesis pathway is important and/or efficient at increasing NAD levels in vivo. In this study, Nmnat3 overexpression in mice efficiently increased NAD levels in various tissues and prevented aging-related declines in NAD levels. We also demonstrated that Nmnat3-overexpressing (Nmnat3 Tg) mice were protected against diet-induced and aging-associated insulin resistance. Moreover, in skeletal muscles of Nmnat3 Tg mice, TCA cycle activity was significantly enhanced, and the energy source for oxidative phosphorylation was shifted toward fatty acid oxidation. Furthermore, reactive oxygen species (ROS) generation was significantly suppressed in aged Nmnat3 Tg mice. Interestingly, we also found that concentrations of the NAD analog nicotinamide guanine dinucleotide (NGD) were dramatically increased in Nmnat3 Tg mice. These results suggest that Nmnat3 overexpression improves metabolic health and that Nmnat3 is an attractive therapeutic target for metabolic disorders that are caused by aging.

It is controversial whether sodium glucose transporter (SGLT) 2 inhibitors increase glucagon secretion via direct inhibition of SGLT2 in pancreatic α cells. The role of SGLT1 in α cells is also unclear. We aimed to elucidate these points that are important not only for basic research but also for clinical insight.

Oxidative stress and mitochondrial dysfunction are important determinants of neurodegeneration in secondary progressive multiple sclerosis (SPMS). We previously showed that febuxostat, a xanthine oxidase inhibitor, ameliorated both relapsing-remitting and secondary progressive experimental autoimmune encephalomyelitis (EAE) by preventing neurodegeneration in mice. In this study, we investigated how febuxostat protects neuron in secondary progressive EAE. A DNA microarray analysis revealed that febuxostat treatment increased the CNS expression of several mitochondria-related genes in EAE mice, most notably including GOT2, which encodes glutamate oxaloacetate transaminase 2 (GOT2). GOT2 is a mitochondrial enzyme that oxidizes glutamate to produce α-ketoglutarate for the Krebs cycle, eventually leading to the production of adenosine triphosphate (ATP). Whereas GOT2 expression was decreased in the spinal cord during the chronic progressive phase of EAE, febuxostat-treated EAE mice showed increased GOT2 expression. Moreover, febuxostat treatment of Neuro2a cells in vitro ameliorated ATP exhaustion induced by rotenone application. The ability of febuxostat to preserve ATP production in the presence of rotenone was significantly reduced by GOT2 siRNA. GOT2-mediated ATP synthesis may be a pivotal mechanism underlying the protective effect of febuxostat against neurodegeneration in EAE. Accordingly, febuxostat may also have clinical utility as a disease-modifying drug in SPMS.

Dysregulation of glucagon is associated with the pathophysiology of type 2 diabetes. We previously reported that postprandial hyperglucagonemia is more obvious than fasting hyperglucagonemia in type 2 diabetes patients. However, which nutrient stimulates glucagon secretion in the diabetic state and the underlying mechanism after nutrient intake are unclear. To answer these questions, we measured plasma glucagon levels in diabetic mice after oral administration of various nutrients. The effects of nutrients on glucagon secretion were assessed using islets isolated from diabetic mice and palmitate-treated islets. In addition, we analyzed the expression levels of branched chain amino acid (BCAA) catabolism-related enzymes and their metabolites in diabetic islets. We found that protein, but not carbohydrate or lipid, increased plasma glucagon levels in diabetic mice. Among amino acids, BCAAs, but not the other essential or nonessential amino acids, increased plasma glucagon levels. BCAAs also directly increased the intracellular calcium concentration in α cells. When BCAAs transport was suppressed by an inhibitor of system L-amino acid transporters, glucagon secretion was reduced even in the presence of BCAAs. We also found that the expression levels of BCAA catabolism-related enzymes and their metabolite contents were altered in diabetic islets and palmitate-treated islets compared to control islets, indicating disordered BCAA catabolism in diabetic islets. Furthermore, BCKDK inhibitor BT2 suppressed BCAA-induced hypersecretion of glucagon in diabetic islets and palmitate-treated islets. Taken together, postprandial hypersecretion of glucagon in the diabetic state is attributable to disordered BCAA catabolism in pancreatic islet cells.

Cyclic adenosine monophosphate responsive element-binding protein-1-regulated transcription coactivator-1 (CRTC1) is a cytoplasmic coactivator that translocates to the nucleus in response to cyclic adenosine monophosphate. Whole-body knockdown of Crtc1 causes obesity, resulting in increased food intake and reduced energy expenditure. CRTC1 is highly expressed in the brain; therefore, it might play an important role in energy metabolism via the neuronal pathway. However, the precise mechanism by which CRTC1 regulates energy metabolism remains unknown. Here, we showed that mice lacking CRTC1, specifically in steroidogenic factor-1 expressing cells (SF1 cells), were sensitive to high-fat diet (HFD)-induced obesity, exhibiting hyperphagia and increased body weight gain. The loss of CRTC1 in SF1 cells impaired glucose metabolism. Unlike whole-body CRTC1 knockout mice, SF1 cell-specific CRTC1 deletion did not affect body weight gain or food intake in normal chow feeding. Thus, CRTC1 in SF1 cells is required for normal appetite regulation in HFD-fed mice. CRTC1 is primarily expressed in the brain. Within the hypothalamus, which plays an important role for appetite regulation, SF1 cells are only found in ventromedial hypothalamus. RNA sequencing analysis of microdissected ventromedial hypothalamus samples revealed that the loss of CRTC1 significantly changed the expression levels of certain genes. Our results revealed the important protective role of CRTC1 in SF1 cells against dietary metabolic imbalance.

Background Limited data exist on the prognostic factors for patients with ischemic stroke and active cancer. Methods and Results We conducted a prospective, multicenter, observational study in Japan, including patients with acute ischemic stroke and active cancer, to investigate the prognostic factors. We followed up the patients for 1 year after stroke onset. The patients were divided into 2 groups according to cryptogenic stroke and known causes (small-vessel occlusion, large-artery atherosclerosis, cardioembolism, and other determined cause), and survival was compared. The hazard ratios (HRs) and 95% CIs for mortality were calculated using Cox regression models. We identified 135 eligible patients (39% women; median age, 75 years). Of these patients, 51% had distant metastasis. A total of 65 (48%) and 70 (52%) patients had cryptogenic stroke and known causes, respectively. Patients with cryptogenic stroke had significantly shorter survival than those with known causes (HR [95% CI], 3.11 [1.82-5.32]). The multivariable Cox regression analysis revealed that distant metastasis, plasma D-dimer levels, venous thromboembolism (either deep venous thrombosis or pulmonary embolism) complications at stroke onset were independent predictors of mortality after adjusting for potential confounders. Cryptogenic stroke was associated with prognosis in univariable analysis but was not significant in multivariable analysis. The plasma D-dimer levels stratified the prognosis of patients with ischemic stroke and active cancer. Conclusions The prognosis of patients with acute ischemic stroke and active cancer varied considerably depending on stroke mechanism, distant metastasis, and coagulation abnormalities. The present study confirmed that coagulation abnormalities were crucial in determining the prognosis of such patients.

Obesity arises from impaired energy balance, which is centrally coordinated by leptin through activation of the long form of leptin receptor (Leprb). Obesity causes central leptin resistance. However, whether enhanced peripheral leptin sensitivity could overcome central leptin resistance remains obscure. A peripheral metabolic organ targeted by leptin is the liver, with low Leprb expression. We here show that mice fed a high-fat diet (HFD) and obese patients with hepatosteatosis exhibit increased expression of hepatic helicase with zinc finger 2, a transcriptional coactivator (Helz2), which functions as a transcriptional coregulator of several nuclear receptors, including peroxisome proliferator-activated receptor γ in vitro. To explore the physiological importance of Helz2, we generated Helz2-deficient mice and analyzed their metabolic phenotypes. Helz2-deficient mice showing hyperleptinemia associated with central leptin resistance were protected against HFD-induced obesity and had significantly up-regulated hepatic Leprb expression. Helz2 deficiency and adenovirus-mediated liver-specific exogenous Leprb overexpression in wild-type mice significantly stimulated hepatic AMP-activated protein kinase on HFD, whereas Helz2-deficient db/db mice lacking functional Leprb did not. Fatty acid-β oxidation was increased in Helz2-deficeint hepatocytes, and Helz2-deficient mice revealed increased oxygen consumption and decreased respiratory quotient in calorimetry analyses. The enhanced hepatic AMP-activated protein kinase energy-sensing pathway in Helz2-deficient mice ameliorated hyperlipidemia, hepatosteatosis, and insulin resistance by reducing lipogenic gene expression and stimulating lipid-burning gene expression in the liver. These findings together demonstrate that Helz2 deficiency ameliorates HFD-induced metabolic abnormalities by stimulating endogenous hepatic Leprb expression, despite central leptin resistance. Hepatic HELZ2 might be a novel target molecule for the treatment of obesity with hepatosteatosis.