Sphingosine 1-phosphate (S1P) is accumulated in lipoproteins, especially high-density lipoprotein (HDL), in plasma. However, it remains uncharacterized how extracellular S1P is produced in the CNS. The treatment of rat astrocytes with retinoic acid and dibutyryl cAMP, which induce apolipoprotein E (apoE) synthesis and HDL-like lipoprotein formation, stimulated extracellular S1P accumulation in the presence of its precursor sphingosine. The released S1P was present together with apoE particles in the HDL fraction. S1P release from astrocytes was inhibited by the treatment of the cells with glybenclamide or small interfering RNAs specific to ATPbinding cassette transporter A1 (ABCA1). Astrocytes from Abca1)/) mice also showed impairment of retinoic acid/dibutyryl cAMP-induced S1P release in association with the blockage of HDL-like lipoprotein formation. However, the formation of either apoE or lipoprotein itself was not sufficient, and additional up-regulation of ABCA1 was requisite to stimulate S1P release. We conclude that the S1P release from astrocytes is coupled with lipoprotein formation through ABCA1. Keywords: apolipoprotein E, astrocyte, ATP-binding cassette transporter A1, high-density lipoprotein, sphingosine 1-phosphate. Sphingosine 1-phosphate (S1P), a sphingolipid metabolite, is a pleiotropic lipid mediator involved in a variety of cell activities, including morphology, motility, and growth. Five subtypes of S1P-specific receptors, S1P 1-5 , have been isolated so far (Ishii et al. 2004). In neural cells, these receptors are expressed in a cell-specific manner (Van Brocklyn et al. 1999;Sato et al. 2000) and involved in the regulation of neural cell functions; for example, a rapid process retraction through S1P 5 in oligodendrocytes (Jaillard et al. 2005), rounding of the cell body in PC12 and N1E115 cells (Postma et al. 1996;Sato et al. 1997) possibly through S1P 2 (Van Brocklyn et al. 1999), and regulation of motility in astrocytes and glioma cells . In addition, S1P induces the proliferation in astroglial cells, which was associated with the activation of extracellular signal-regulated kinase (ERK), activation of the phospholipase C, and Ca 2+ mobilization (Tas and Koschel 1998;Sato et al. 1999;Pebay et al. 2001). S1P 1 is more important than S1P 2 for the stimulation of ERK, while S1P 2 may be responsible for the activation of phospholipase C and Ca 2+ mobilization (Sato et al. 2000;Malchinkhuu et al. 2003). Thus, S1P and its receptors may play important roles in maintaining the functions of the CNS.We have recently demonstrated that S1P is concentrated in lipoproteins, such as high-density lipoprotein (HDL), in circulating blood (Murata et al. 2000b), and that the lipid mediates lipoprotein-induced anti-atherogenic actions, including cell survival, cell migration, and inhibition of adhesion molecule expression through S1P receptors in endothelial cells Okajima 2002). Thus, lipoproteins seem to serve as carriers for extracellular S1P in circulating blood. In astrocytes as well, plasma HDL ...

A potential role for 1-oleoyl-sn-glycero-3-phosphate or lysophosphatidic acid (LPA) and sphingosine-1-phosphate (S1P) in the regulation of malignant diseases has been widely considered. In this study, we found that in transformed astroglial cells, the expression profile of lysophospholipid receptor mRNA and the action modes of LPA and S1P on cell motility were changed: there was a change in the acquisition of the ability of LPA to stimulate cell migration and a change in the migratory response to S1P from stimulation through S1P 1 to inhibition through S1P 2 . LPA-induced cell migration was almost completely inhibited by either pertussis toxin, LPA 1 receptor antagonists including Ki16425 (3-(4-[4-([1-(2-chlorophenyl)ethoxy]carbonyl amino)-3-methyl-5-isoxazolyl] benzylsulfonyl)propanoic acid) or an inhibitor of phosphatidylinositol 3-kinase (PI3K) wortmannin. The LPA-induced action was also suppressed, although incompletely, by several specific inhibitors for intracellular signaling pathways including Rac1, Cdc42, p38 mitogen-activated protein kinase (p38MAPK) and c-Jun terminal kinase (JNK), but not extracellular signalregulated kinase. Nearly complete inhibition of migration response to LPA, however, required simultaneous inhibition of both the p38MAPK and JNK pathways. Inhibition of Rac1 suppressed JNK but not p38MAPK, while the activity of p38MAPK was abolished by a dominantnegative form of Cdc42. These findings suggest that, in glioma cells, the PI3K/Cdc42/p38MAPK and PI3K/ Rac1/JNK pathways are equally important for LPA 1 receptor-mediated migration.

Increase in the concentration of plasma L-cysteine is closely associated with defective insulin secretion from pancreatic β-cells, which results in type 2 diabetes (T2D). In this study, we investigated the effects of prolonged L-cysteine treatment on glucosestimulated insulin secretion (GSIS) from mouse insulinoma 6 (MIN6) cells and from mouse pancreatic islets, and found that the treatment reversibly inhibited glucose-induced ATP production and resulting GSIS without affecting proinsulin and insulin synthesis. Comprehensive metabolic analyses using capillary electrophoresis time-of-flight mass spectrometry showed that prolonged L-cysteine treatment decreased the levels of pyruvate and its downstream metabolites. In addition, methyl pyruvate, a membrane-permeable form of pyruvate, rescued L-cysteine-induced inhibition of GSIS. Based on these results, we found that both in vitro and in MIN6 cells, L-cysteine specifically inhibited the activity of pyruvate kinase muscle isoform 2 (PKM2), an isoform of pyruvate kinases that catalyze the conversion of phosphoenolpyruvate to pyruvate. L-cysteine also induced PKM2 subunit dissociation (tetramers to dimers/monomers) in cells, which resulted in impaired glucose-induced ATP production for GSIS. DASA-10 (NCGC00181061, a substituted N,N′-diarylsulfonamide), a specific activator for PKM2, restored the tetramer formation and the activity of PKM2, glucoseinduced ATP production, and biphasic insulin secretion in L-cysteinetreated cells. Collectively, our results demonstrate that impaired insulin secretion due to exposure to L-cysteine resulted from its direct binding and inactivation of PKM2 and suggest that PKM2 is a potential therapeutic target for T2D.A metabolite, L-cysteine, is found in blood plasma, and its concentration is closely associated with an increase in fat mass and the body-mass index. These values are used as an index of obesity (1, 2), which is a major risk factor for type 2 diabetes (T2D) (3). The relationship between L-cysteine and diabetes has attracted attention because there is increasing evidence for a positive correlation between increases in plasma L-cysteine concentrations and the development and progression of diabetes. For example, increased plasma L-cysteine concentrations were associated with T2D in African American women (4), renal insufficiency [reduced glomerular filtration rate (GFR)] in T2D patients (5), obstructive sleep apnea [a risk factor for diabetes (6, 7)], and insulin resistance among Europeans (8).Reduced insulin secretion from pancreatic β-cells is the major cause of T2D (9, 10). Many investigators have studied the molecular mechanisms of glucose-stimulated insulin secretion (GSIS), which have been elucidated in detail. Elevated extracellular glucose concentration results in the enhancement of ATP production, an increased ATP/ADP ratio, the closure of ATP-sensitive K channels (K ATP channels), and depolarization (11). The resulting activation of voltage-dependent Ca 2+ channels (VDCCs) induces an influx of calcium ions and elevated...

In this study, to elucidate the mechanisms of oocyte hydration in the Japanese eel (Anguilla japonica), we examined the in vivo and in vitro morphological changes and hydration process occurring during oocyte maturation and ovulation. We also investigated the effects of the presence of ovarian follicles, aquaporin water permeability (HgCl(2)), and yolk proteolysis (bafilomycin A1) inhibitors, gap junction uncouplers (carbenoxolone and 1-octanol), a 3beta-hydroxysteroid dehydrogenase inhibitor (trilostane), and a P450 antagonist (aminoglutethimide) on oocyte hydration during in vitro oocyte maturation and ovulation. The oocytes underwent more than threefold increase in volume during maturation and ovulation, which was artificially induced by injecting salmon pituitary extracts and 17,20beta-dihydroxy-4-pregnen-3-one (DHP). Wet and dry weight measurements indicated that water accumulation during oocyte maturation is the major factor contributing to the follicular diameter increase, suggesting that follicular diameter measurements can be used as a hydration index. In the in vitro experiments, human chorionic gonadotropin (HCG) and DHP caused an increase in the diameter of follicle-enclosed oocytes but not defolliculated oocytes. Addition of HgCl(2) and bafilomycin A1 to the incubation media inhibited the HCG- and DHP-induced increase in the follicular diameter in a dose-dependent manner. Neither carbenoxolone nor 1-octanol influenced the HCG-induced increase in the follicular diameter. Trilostane and aminoglutethimide slightly but significantly inhibited HCG-induced oocyte hydration. Consequently, we concluded that ovarian follicles are essential for HCG- and DHP-induced oocyte hydration. Furthermore, aquaporin facilitates water uptake by acting as a water channel, and yolk proteolysis is essential for water influx into oocytes via osmotic mechanisms.

Background: Although Clara cell secretory protein (CC-10) has been ascribed an anti-inflammatory role in lung diseases, its precise role remains unclear. Objective: To further our understanding of the role of CC-10 in inflammatory lung diseases, CC-10 protein levels were measured. Methods: Sera or bronchoalveolar lavage (BAL) fluids were collected from patients with different inflammatory lung diseases including bronchial asthma, chronic obstructive lung disease (COPD), sarcoidosis, idiopathic interstitial pneumonia (IIP), chronic eosinophilic pneumonia (CEP), pneumonia and lung cancer. Serum CC-10 concentrations were measured by enzyme-linked immunosorbent assay using urinary protein-1 antibody. Then, the relationships between CC-10 concentrations and lung diseases were investigated. Immunohistochemistry was performed using lung biopsy samples. Results: Increased serum CC-10 levels were recognized in IIP patients, while CC-10 levels were decreased in bronchial asthma patients and CEP patients. Immunohistochemistry revealed an aberrant expression in areas of fibrosis in IIP patients. Serum CC-10 concentrations were not associated with severity among IIP, COPD, and sarcoidosis. In contrast, serum CC-10 concentrations were correlated with FEV₁/FVC in bronchial asthma patients. Conclusions: Although the number of patients was quite limited, these data provide new insights into the role of CC-10 in lung diseases, and the possibility that the CC-10 concentration in serum could be a new marker indicating the severity of bronchial asthma.

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Immunoregulatory conditions in atopic dermatitis (AD) involve both Th1 and Th2, although primarily Th2. The suppressor of cytokine signalling 3 (SOCS3) may mediate Th2 regulatory response through negative regulation of Th1 pathways. We examined SOCS3 expression in skin lesions of patients with severe AD and compared it with that in patients with the Th1 disorder, psoriasis. We assessed SOCS3 expression in skin lesions from seven patients with severe AD and five patients with typical psoriasis, and in skin from three normal subjects, using immunohistochemical staining, in situ hybridization and reverse transcription PCR. Strong SOCS3 expression was found in skin lesions in all patients with AD, but only weak expression in patients with psoriasis and in normal subjects. Overexpression of SOCS3 is prominent in the skin of patients with severe AD and consequently may prove useful for evaluating the severity of AD as a Th2-dominant condition.