Taurine down-regulates basal and osmolarity-induced gene expression of its transporter, but not the gene expression of its biosynthetic enzymes, in astrocyte primary cultures

Taurine content of astrocytes is primarily regulated by transport from the extracellular medium and endogenous biosynthesis from cysteine. We have investigated the gene expression of the taurine transporter (TauT) and the taurine biosynthetic enzymes, cysteine dioxygenase (CDO) and cysteine sulfinate decarboxylase (CSD), in astrocyte primary cultures in relationship to cell taurine content. TauT, CDO, and CSD mRNA levels were determined through quantitative RT-PCR. Cell taurine content was depleted by adapting the cells to a taurine-free chemically defined medium and increased by incubating the cells in the same medium containing exogenous taurine. With increased cell taurine content the level of TauT mRNA decreased, whereas the levels of CDO and CSD mRNA remained unchanged. In astrocytes exposed to a hyperosmotic medium the TauT mRNA level increased, whereas the CDO and CSD mRNA levels were not significantly altered. The osmolarity-induced up-regulation of TauT mRNA expression was fully prevented by increasing cell taurine content. Thus, the gene expression of the taurine transporter, but not that of the taurine biosynthetic enzymes, appears to be under the control of two antagonistic regulations, namely, a taurine-induced down-regulation and an osmolarity-induced up-regulation.     

Enhancement of menadione stress tolerance in yeast by accumulation of hypotaurine and taurine: co-expression of cDNA clones, from Cyprinus carpio, for cysteine dioxygenase and cysteine sulfinate decarboxylase in Saccharomyces cerevisiae

Taurine is known to function as a protectant against various stresses in animal cells. In order to utilize taurine as a compatible solute for stress tolerance of yeast, isolation of cDNA clones for genes encoding enzymes involved in biosynthesis of taurine was attempted. Two types of cDNA clones corresponding to genes encoding cysteine dioxygenase (CDO1 and CDO2) and a cDNA clone for cysteine sulfinate decarboxylase (CSD) were isolated from Cyprinus carpio. Deduced amino acid sequences of the two CDOs and that of CSD showed high similarity to those of CDOs and those of CSDs from other organisms, respectively. The coding regions of CDO1, CDO2, and CSD were subcloned into an expression vector, pESC-TRP, for Saccharomyces cerevisiae. Furthermore, to enhance the efficiency of synthesis of taurine in S. cerevisiae, a CDO–CSD fusion was designed and expressed. Expression of CDO and CSD proteins, or the CDO–CSD fusion protein was confirmed by Western blot analysis. HPLC analysis showed that the expression of the proteins led to enhancement of the accumulation level of hypotaurine, a precursor of taurine, rather than taurine. The yeast cells expressing corresponding genes showed tolerance to oxidative stress induced by menadione, but not to freezing–thawing stress.     

Physiological significance of the taurine transporter and taurine biosynthetic enzymes in 3T3-L1 adipocytes

The intracellular level of taurine is maintained both by the taurine transporter (TAUT) and by endogenous synthesis from Met and Cys. We investigated in the present study the regulation of TAUT and of cysteine dioxygenase (CDO), one of the major taurine biosynthetic enzymes, in 3T3-L1 adipocytes. The TAUT activity, expression of TAUT and CDO mRNA were up-regulated by hypertonicity. In contrast, the TAUT activity, expression of TAUT and CDO mRNA were down-regulated by taurine-rich conditions. Furthermore, it was indicated that the up-regulation of TAUT activity resulted from the increased number of expressed TAUT, and not by the change in affinity of TAUT. On the other hand, the taurine-induced down-regulation of TAUT activity resulted not only from a decrease in the number of expressed TAUT but also from a decrease in their affinity. These results suggest that murine TAUT and CDO were cooperatively regulated in response to hypertonicity and taurine-rich conditions.     

The synthesis and role of taurine in the Japanese eel testis

In teleost fish, the progestin 17α, 20β-dihydroxy-4-pregnen-3-one (DHP) is an essential component of the spermatogenesis pathway. In a series of investigations on the mechanisms underlying progestin-stimulated spermatogenesis, we have found that DHP up-regulates the expression of cysteine dioxygenase1 (CDO1) in the Japanese eel testis. CDO1 is one of the enzymes involved in the taurine biosynthesis pathway. To evaluate whether taurine is synthesized in the eel testis, cysteine sulfinate decarboxylase (CSD), another enzyme involved in taurine synthesis, was isolated from this species. RT-PCR and in vitro eel testicular culture revealed that although CSD was also expressed in eel testis, neither DHP nor other sex steroids affect CSD mRNA expression in a similar manner to CDO1. Using an in vitro eel testicular culture system, we further investigated the effects of DHP on taurine synthesis in the eel testis. HPLC analysis showed that DHP treatment significantly increases the taurine levels in the eel testis. These results suggest that DHP promotes taurine synthesis via the up-regulation of CDO1 mRNA expression during eel spermatogenesis. Furthermore, we observed from our analysis that although taurine does not induce complete spermatogenesis, it promotes spermatogonial DNA synthesis and the expression of Spo11, a meiosis-specific marker. These data thus suggest that taurine augments the effects of sex steroids in the promotion of spermatogonial proliferation and/or meiosis and hence that taurine plays important roles in spermatogenesis.     

Extrahepatic tissues compensate for loss of hepatic taurine synthesis in mice with liver-specific knockout of cysteine dioxygenase

Because hepatic cysteine dioxygenase (CDO) appears to play the major role in controlling cysteine catabolism in the intact rat, we characterized the effect of a lack of hepatic CDO on the regulation of cysteine and its metabolites at the whole body level. In mice with liver-specific deletion of CDO expression, hepatic and plasma cysteine levels increased. In addition, in mice with liver-specific deletion of CDO expression, the abundance of CDO and the proportion of CDO existing as the mature, more active isoform increased in extrahepatic tissues that express CDO (kidney, brown fat, and gonadal fat). CDO abundance was also increased in the pancreas, where most of the enzyme in both control and liver CDO-knockout mice was in the more active isoform. This upregulation of CDO concentration and active-site cofactor formation were not associated with an increase in CDO mRNA and thus presumably were due to a decrease in CDO degradation and an increase in CDO cofactor formation in association with increased exposure of extrahepatic tissues to cysteine in mice lacking hepatic CDO. Extrahepatic tissues of liver CDO-knockout mice also had higher levels of hypotaurine, consistent with increased metabolism of cysteine by the CDO/cysteinesulfinate decarboxylase pathway. The hepatic CDO-knockout mice were able to maintain normal levels of glutathione, taurine, and sulfate. The maintenance of taurine concentrations in liver as well as in extrahepatic tissues is particularly notable, since mice were fed a taurine-free diet and liver is normally considered the major site of taurine biosynthesis. This redundant capacity for regulation of cysteine concentrations and production of hypotaurine/taurine is additional support for the body's robust mechanisms for control of body cysteine levels and indicates that extrahepatic tissues are able to compensate for a lack of hepatic capacity for cysteine catabolism.     

Role of the Liver in Regulation of Body Cysteine and Taurine Levels: A Brief Review

The first-pass metabolism of dietary sulfur amino acids by the liver and the robust upregulation of hepatic cysteine dioxygenase activity in response to an increase in dietary protein or sulfur amino acid level gives the liver a primary role in the removal of excess cysteine and in the synthesis of taurine. Hepatic taurine synthesis is largely restricted by the low availability of cysteinesulfinate as substrate for cysteinesulfinate decarboxylase, and taurine production is increased when cysteinesulfinate increases in response to an increase in the hepatic cysteine concentration and the associated increase in cysteine dioxygenase activity. The upregulation of cysteine dioxygenase in the presence of cysteine is a consequence of diminished ubiquitination of cysteine dioxygenase and a slower rate of degradation by the 26S proteasome.     

Taurine deficiency,synthesis and transport in the mdx mouse model for Duchenne Muscular Dystrophy

The amino acid taurine is essential for the function of skeletal muscle and administration is proposed as a treatment for Duchenne Muscular Dystrophy (DMD). Taurine homeostasis is dependent on multiple processes including absorption of taurine from food, endogenous synthesis from cysteine and reabsorption in the kidney. This study investigates the cause of reported taurine deficiency in the dystrophic mdx mouse model of DMD. Levels of metabolites (taurine, cysteine, cysteine sulfinate and hypotaurine) and proteins (taurine transporter [TauT], cysteine deoxygenase and cysteine sulfinate dehydrogenase) were quantified in juvenile control C57 and dystrophic mdx mice aged 18 days, 4 and 6 weeks. In C57 mice, taurine content was much higher in both liver and plasma at 18 days, and both cysteine and cysteine deoxygenase were increased. As taurine levels decreased in maturing C57 mice, there was increased transport (reabsorption) of taurine in the kidney and muscle. In mdx mice, taurine and cysteine levels were much lower in liver and plasma at 18 days, and in muscle cysteine was low at 18 days, whereas taurine was lower at 4: these changes were associated with perturbations in taurine transport in liver, kidney and muscle and altered metabolism in liver and kidney. These data suggest that the maintenance of adequate body taurine relies on sufficient dietary intake of taurine and cysteine availability and metabolism, as well as retention of taurine by the kidney. This research indicates dystrophin deficiency not only perturbs taurine metabolism in the muscle but also affects taurine metabolism in the liver and kidney, and supports targeting cysteine and taurine deficiency as a potential therapy for DMD.     

Effects of Taurine on Glial Cells Apoptosis and Taurine Transporter Expression in Retina Under Diabetic Conditions

Taurine, a ß-aminosulfonic acid, has been reported to reduce the risk of a number of diseases, including cardiovascular disease, diabetes, and also perhaps to reduce neurodegeneration in the elderly. The transport of taurine is known to be mediated by taurine transporter (TauT). The purpose of this study is to examine the effects of taurine on glial cells apoptosis and on TauT expression in retina of diabetic rats and retinal glial cells cultured with high glucose. TdT-mediated dUTP-biotin nick-end labeling (TUNEL) staining analysis showed that the number of TUNEL-positive cells in taurine treated diabetic rats was significantly lower than those of untreated diabetic rats over the 8-, and 12-week time courses, respectively (all P < 0.001). No TUNEL-positive cells were observed in retina of control groups and taurine treated control groups. In cultured retinal glial cells, the apoptosis in high glucose-treated cells was significantly increased vs the control. When the cells were incubated with high glucose and taurine at 0.1, 1.0 and 10 mmol/l, the percentage of apoptosis was significantly decreased to 16.4, 5.7 and 7.6% respectively (all P < 0.05). With supplementation of taurine in diet and culture medium, higher expression of TauT in retina of diabetic rats and cultured retinal glial cells under diabetic conditions were detected by western-blotting (P < 0.05). Taken together, our data suggest that diabetes or high glucose induced retinal glial cells apoptosis can be inhibited by taurine, and that taurine reverses the diabetes-induced or high glucose-induced decrease in TauT expression.     

High expression of the taurine transporter TauT in primary cilia of NIH3T3 fibroblasts

Taurine, present in high concentrations in various mammalian cells, is essential for regulation of cell volume, cellular oxidative status as well as the cellular Ca2+ homeostasis. Cellular taurine content is a balance between active uptake through the saturable, Na(+)-dependent taurine transporter TauT, and passive release via a volume-sensitive leak pathway. Here we demonstrate that: (i) TauT localizes to the primary cilium of growth-arrested NIH3T3 fibroblasts, (ii) long-term exposure to TNF(alpha) or hypertonic sucrose medium, i.e., growth medium supplemented with 100 mM sucrose, increases ciliary TauT expression and (iii) long-term exposure to hypertonic taurine medium, i.e., growth medium supplemented with 100 mM taurine, reduces ciliary TauT expression. These results point to an important role of taurine in the regulation of physiological processes located to the primary cilium.     

Regulation of the Cellular Content of the Organic Osmolyte Taurine in Mammalian Cells

Change in the intracellular concentration of osmolytes or the extracellular tonicity results in a rapid transmembrane water flow in mammalian cells until intracellular and extracellular tonicities are equilibrated. Most cells respond to the osmotic cell swelling by activation of volume-sensitive flux pathways for ions and organic osmolytes to restore their original cell volume. Taurine is an important organic osmolyte in mammalian cells, and taurine release via a volume-sensitive taurine efflux pathway is increased and the active taurine uptake via the taurine specific taurine transporter TauT decreased following osmotic cell swelling. The cellular signaling cascades, the second messengers profile, the activation of specific transporters, and the subsequent time course for the readjustment of the cellular content of osmolytes and volume vary from cell type to cell type. Using Ehrlich ascites tumor cells, NIH3T3 mouse fibroblasts and HeLa cells as biological systems, it is revealed that phospholipase A₂-mediated mobilization of arachidonic acid from phospholipids and subsequent oxidation of the fatty acid via lipoxygenase systems to potent eicosanoids are essential elements in the signaling cascade that is activated by cell swelling and leads to release of osmolytes. The cellular signaling cascade and the activity of the volume-sensitive taurine efflux pathway are modulated by elements of the cytoskeleton, protein tyrosine kinases/phosphatases, GTP-binding proteins, Ca²⁺/calmodulin, and reactive oxygen species and nucleotides. Serine/threonine phosphorylation of the active taurine uptake system TauT or a putative regulator, as well as change in the membrane potential, are important elements in the regulation of TauT activity. A model describing the cellular sequence, which is activated by cell swelling and leads to activation of the volume-sensitive efflux pathway, is presented at the end of the review.     

Taurine transporter gene expression in peripheral mononuclear blood cells of type 2 diabetic patients

Taurine acts as antioxidant, cell osmolyte, modulator of glucose metabolism, and plays a role in the retinal function. It is 10³-fold more concentrated in the intracellular than in the extracellular milieu due to a specific taurine-Na-dependent transporter (TauT), which is upregulated by hypertonicity, low extracellular taurine, or oxidative stress and acutely downregulated ‘in vitro’ by high glucose concentrations. Aim of this study was to investigate whether TauT expression was modified in mononuclear peripheral blood cells (MPC) of type 2 diabetic patients with or without micro/macrovascular complications. Plasma taurine, as well as other sulphur-containing aminoacids (assayed by HPLC) and TauT gene expression (assayed by real-time PCR analysis) were measured in MPC of 45 controls and of 81 age-and-sex matched type 2 diabetic patients with or without micro/macrovascular complications. Median value (interquartile range) of plasma taurine was significantly lower in diabetic patients than in controls [28.7 (13.7) μmol/l vs. 46.5 (20.3) μmol/l; P?<?0.05], while median TauT expression, in arbitrary units, was significantly higher in diabetics than in controls [3.8 (3.9) vs. 1 (1.3); P?<?0.05) and was related to HbA1c only in controls (r?=?0.34; P?<?0.05). Patients with retinopathy (n?=?25) had lower TauT expression than those who were unaffected [3.1 (2.8) vs. 4.1 (3.4); P?<?0.05], while persistent micro/macroalbuminuria was associated with unchanged TauT expression. A trend toward reduction in TauT expression was observed in patients with macroangiopathy [n?=?27; 3.3 (2.5) vs. 4 [3.7]; P?=?NS]. In conclusion, TauT gene is overexpressed in MPC of type 2 diabetic patients, while presence of retinopathy is specifically associated with a drop in TauT overexpression, suggesting its possible involvement in this microangiopathic lesion.     

Molecular characterization and expression pattern of taurine transporter in zebrafish during embryogenesis

Taurine and its transporter (TauT) are expressed in preimplantation embryos, but their role in embryogenesis is not known. To investigate the role of TauT during embryonic development, we cloned and functionally characterized the zebrafish TauT. The zebrafish TauT cDNA codes for a protein of 625 amino acids which is highly homologous to mammalian TauT. When expressed in mammalian cells, zebrafish TauT mediates taurine uptake in a Na(+)/Cl(-)-dependent manner with a Na(+):Cl(-):taurine stoichiometry of 2:1:1. In the zebrafish embryo, taurine and TauT mRNA are present during early cleavage stages, indicating that both the transporter and its substrate are maternally derived. During embryogenesis, zygotic expression of TauT mRNA is evident in the retina, brain, heart, kidney, and blood vessels. Knockdown of TauT by antisense morpholino oligonucleotides leads to cell death in the central nervous system and increased mortality. These findings suggest a specific role for TauT during development in vertebrates.     

Production and Characterization of a New Specific Antiserum Against the Taurine Putative Biosynthetic Enzyme Cysteine Sulfinate Decarboxylase

Abstract: We have shown previously that cysteine sulfinate decarboxylase (CSD), the putative biosynthetic enzyme of taurine in the brain, is identical to the liver enzyme according to biochemical, kinetic, and immunochemical criteria. In the present work, CSD was purified in its native form from rat liver. The purification was performed in eight steps, which included conventional chromatography (diethylaminoethyl cellulose, hydroxylapatite), followed by HPLC (hydrophobic, adsorption, and ion-exchange HPLC). The purification factor was 11,000, and the final yield was around 2%. The procedure led to the enrichment of a protein, the molecular mass of which was 51,000 daltons as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The final fraction was more than 90% homogeneous. By using this fraction as the antigen, an antiserum was raised in rabbit that (a) quantitatively immunoprecipitated CSD activity from liver and brain extract, and (b) immunolabeled one band (51,000 daltons) on immunoblots of partially purified fractions from liver. Enrichment of CSD specific activity and that of the protein immunolabeled by the antiserum for a given step, e.g., hydrophobic HPLC, were consistently parallel. The antiserum was used to carry out CSD immunocytochemistry in cerebellum. Numerous small cells were labeled in the Purkinje cell layer, the granular layer, and the white matter. In the molecular layer, Bergmann radial fibers were im munostained. The Purkinje and stellate cells were devoid of any labeling at the cell body and terminal levels. The antiserum appears to be specific for CSD and suitable for immunocytochemical visualization of CSD in the brain.     

Taurine biosynthesis by neurons and astrocytes

The physiological roles of taurine, a product of cysteine degradation and one of the most abundant amino acids in the body, remain elusive. Taurine deficiency leads to heart dysfunction, brain development abnormalities, retinal degradation, and other pathologies. The taurine synthetic pathway is proposed to be incomplete in astrocytes and neurons, and metabolic cooperation between these cell types is reportedly needed to complete the pathway. In this study, we analyzed taurine synthesis capability as reported by incorporation of radioactivity from [(35)S]cysteine into taurine, in primary murine astrocytes and neurons, and in several transformed cell lines (human (SH-SY5Y) and murine (N1E-115) neuroblastoma, human astrocytoma (U-87 MG and 1321 N1), and rat glioma (C6)). Extensive incorporation of radioactivity from [(35)S]cysteine into taurine was observed in rat glioma cells as well as in primary mouse astrocytes and neurons, establishing the presence of an intact taurine synthesis pathway in these cells. Interestingly, exposure of cells to cysteine or cysteamine resulted in elevated intracellular hypotaurine without a corresponding increase in taurine levels, suggesting that oxidation of hypotaurine limits taurine synthesis in cells. Consistent with its role as an organic osmolyte, taurine synthesis was stimulated under hypertonic conditions in neurons.     

Taurine synthesis and cysteine metabolism in cultured rat astrocytes: effects of hyperosmotic exposure

We investigated mechanisms controlling taurinesynthesis in cultured rat cerebral astrocytes. The mean ± SE rateof taurine synthesis from extracellular cysteine was 21.2 ± 2.0 pmol · mg protein¹ · min¹,whereas taurine degradation was <1.3% of this rate. Eliminating cellular glutathione and inhibiting glutathione biosynthesis increased taurine synthesis from extracellular cysteine by 39%. In cell homogenates, cysteine dioxygenase (CDO) and cysteine-sulfinate decarboxylase activities were 2.4 ± 0.2 and 8.3 ± 2.8 nmol · mg protein¹ · min¹,respectively. CDO activity was strongly dependent on cysteine concentration over physiological and pathophysiological ranges ofintracellular cysteine concentration. Growth in hyperosmotic mediumcaused a greater increase in culture medium taurine content than thatmeasured from cells in isosmotic growth medium. Hyperosmotic treatmenttransiently increased the rate of cysteine accumulation and cellularcysteine and glutathione contents but had no effect on the synthesisrate of taurine from extracellular cysteine. Thus cysteine isaccumulated and then metabolized to taurine through CDO, whose activitydepends on the intracellular cysteine concentration and appears to berate limiting for taurine synthesis. Hyperosmotic exposure increasesnet taurine production yet has no effect on taurine synthesis fromexogenously applied cysteine. Availability of substrate fromintracellular pools must contribute to maintenance of highintracellular taurine during hyperosmotic exposure.

     

Cysteine regulates expression of cysteine dioxygenase and gamma-glutamylcysteine synthetase in cultured rat hepatocytes

Rat hepatocytes cultured for 3 days in basal medium expressed low levels of cysteine dioxygenase (CDO) and high levels of gamma-glutamylcysteine synthetase (GCS). When the medium was supplemented with 2 mmol/l methionine or cysteine, CDO activity and CDO protein increased by >10-fold and CDO mRNA increased by 1.5- or 3.2-fold. In contrast, GCS activity decreased to 51 or 29% of basal, GCS heavy subunit (GCS-HS) protein decreased to 89 or 58% of basal, and GCS mRNA decreased to 79 or 37% of basal for methionine or cysteine supplementation, respectively. Supplementation with cysteine consistently yielded responses of greater magnitude than did supplementation with an equimolar amount of methionine. Addition of propargylglycine to inhibit cystathionine gamma-lyase activity and, hence, cysteine formation from methionine prevented the effects of methionine, but not those of cysteine, on CDO and GCS expression. Addition of buthionine sulfoximine to inhibit GCS, and thus block glutathione synthesis from cysteine, did not alter the ability of methionine or cysteine to increase CDO. GSH concentration was not correlated with changes in either CDO or GCS-HS expression. The effectiveness of cysteine was equivalent to or greater than that of its precursors (S-adenosylmethionine, cystathionine, homocysteine) or metabolites (taurine, sulfate). Taken together, these results suggest that cysteine itself is an important cellular signal for upregulation of CDO and downregulation of GCS.     

Regulation of the expression and subcellular localization of the taurine transporter TauT in mouse NIH3T3 fibroblasts.

The cellular level of the organic osmolyte taurine is a balance between active uptake and passive leak via a volume sensitive pathway. Here, we demonstrate that NIH3T3 mouse fibroblasts express a saturable, high affinity taurine transporter (TauT, Km = 18 microm), and that taurine uptake via TauT is a Na+- and Cl(-)-dependent process with an apparent 2.5 : 1 : 1 Na+/Cl-/taurine stoichiometry. Transport activity is reduced following acute administration of H2O2 or activators of protein kinases A or C. TauT transport activity, expression and nuclear localization are significantly increased upon serum starvation (24 h), exposure to tumour necrosis factor alpha (TNFalpha; 16 h), or hyperosmotic medium (24 h); conditions that are also associated with increased localization of TauT to the cytosolic network of microtubules. Conversely, transport activity, expression and nuclear localization of TauT are reduced in a reversible manner following long-term exposure (24 h) to high extracellular taurine concentration. In contrast to active taurine uptake, swelling-induced taurine release is significantly reduced following preincubation with TNFalpha (16 h) but unaffected by high extracellular taurine concentration (24 h). Thus, in NIH3T3 cells, (a) active taurine uptake reflects TauT expression; (b) TauT activity is modulated by multiple stimuli, both acutely, and at the level of TauT expression; (c) the subcellular localization of TauT is regulated; and (d) volume-sensitive taurine release is not mediated by TauT.