Increasing of blastocyst rate and gene expression in co-culture of bovine embryos with adult adipose tissue-derived mesenchymal stem cells.

PURPOSE: Despite advances in the composition of defined embryo culture media, co-culture with somatic cells is still used for bovine in vitro embryo production (IVEP) in many laboratories worldwide. Granulosa cells are most often used for this purpose, although recent work suggests that co-culture with stem cells of adult or embryonic origin or their derived biomaterials may improve mouse, cattle, and pig embryo development. MATERIALS AND METHODS: In experiment 1, in vitro produced bovine embryos were co-cultured in the presence of two concentrations of bovine adipose tissue-derived mesenchymal cells (b-ATMSCs; 103 and 104 cells/mL), in b-ATMSC preconditioned medium (SOF-Cond), or SOF alone (control). In experiment 2, co-culture with 104 b-ATMSCs/mL was compared to the traditional granulosa cell co-culture system (Gran). RESULTS: In experiment 1, co-culture with 104 b-ATMSCs/mL improved blastocyst rates in comparison to conditioned and control media (p < 0.05). Despite that it did not show difference with 103 b-ATMSCs/mL (p = 0.051), group 104 b-ATMSCs/mL yielded higher results of blastocyst production. In experiment 2, when compared to group Gran, co-culture with 104 b-ATMSCs/mL improved not only blastocyst rates but also quality as assessed by increased total cell numbers and mRNA expression levels for POU5F1 and G6PDH (p < 0.05). CONCLUSIONS: Co-culture of bovine embryos with b-ATMSCs was more beneficial than the traditional co-culture system with granulosa cells. We speculate that the microenvironmental modulatory potential of MSCs, by means of soluble substances and exosome secretions, could be responsible for the positive effects observed. Further experiments must be done to evaluate if this beneficial effect in vitro also translates to an increase in offspring following embryo transfer. Moreover, this study provides an interesting platform to study the basic requirements during preimplantation embryo development, which, in turn, may aid the improvement of embryo culture protocols in bovine and other species.

Cytokinins and Expression of SWEET, SUT, CWINV and AAP Genes Increase as Pea Seeds Germinate.

Transporter genes and cytokinins are key targets for crop improvement. These genes are active during the development of the seed and its establishment as a strong sink. However, during germination, the seed transitions to being a source for the developing root and shoot. To determine if the sucrose transporter (SUT), amino acid permease (AAP), Sugar Will Eventually be Exported Transporter (SWEET), cell wall invertase (CWINV), cytokinin biosynthesis (IPT), activation (LOG) and degradation (CKX) gene family members are involved in both the sink and source activities of seeds, we used RT-qPCR to determine the expression of multiple gene family members, and LC-MS/MS to ascertain endogenous cytokinin levels in germinating Pisum sativum L. We show that genes that are actively expressed when the seed is a strong sink during its development, are also expressed when the seed is in the reverse role of being an active source during germination and early seedling growth. Cytokinins were detected in the imbibing seeds and were actively biosynthesised during germination. We conclude that, when the above gene family members are targeted for seed yield improvement, a downstream effect on subsequent seed germination or seedling vigour must be taken into consideration.

AraR, an l-Arabinose-Responsive Transcriptional Regulator in Corynebacterium glutamicum ATCC 31831, Exerts Different Degrees of Repression Depending on the Location of Its Binding Sites within the Three Target Promoter Regions.

UNLABELLED: In Corynebacterium glutamicum ATCC 31831, a LacI-type transcriptional regulator AraR, represses the expression of l-arabinose catabolism (araBDA), uptake (araE), and the regulator (araR) genes clustered on the chromosome. AraR binds to three sites: one (BSB) between the divergent operons (araBDA and galM-araR) and two (BSE1 and BSE2) upstream of araE. L-Arabinose acts as an inducer of the AraR-mediated regulation. Here, we examined the roles of these AraR-binding sites in the expression of the AraR regulon. BSB mutation resulted in derepression of both araBDA and galM-araR operons. The effects of BSE1 and/or BSE2 mutation on araE expression revealed that the two sites independently function as the cis elements, but BSE1 plays the primary role. However, AraR was shown to bind to these sites with almost the same affinity in vitro. Taken together, the expression of araBDA and araE is strongly repressed by binding of AraR to a single site immediately downstream of the respective transcriptional start sites, whereas the binding site overlapping the -10 or -35 region of the galM-araR and araE promoters is less effective in repression. Furthermore, downregulation of araBDA and araE dependent on l-arabinose catabolism observed in the BSB mutant and the AraR-independent araR promoter identified within galM-araR add complexity to regulation of the AraR regulon derepressed by L-arabinose. IMPORTANCE: Corynebacterium glutamicum has a long history as an industrial workhorse for large-scale production of amino acids. An important aspect of industrial microorganisms is the utilization of the broad range of sugars for cell growth and production process. Most C. glutamicum strains are unable to use a pentose sugar L-arabinose as a carbon source. However, genes for L-arabinose utilization and its regulation have been recently identified in C. glutamicum ATCC 31831. This study elucidates the roles of the multiple binding sites of the transcriptional repressor AraR in the derepression by L-arabinose and thereby highlights the complex regulatory feedback loops in combination with l-arabinose catabolism-dependent repression of the AraR regulon in an AraR-independent manner.

Endocytosis and vacuolar degradation of the yeast cell surface glucose sensors Rgt2 and Snf3.

Sensing and signaling the presence of extracellular glucose is crucial for the yeast Saccharomyces cerevisiae because of its fermentative metabolism, characterized by high glucose flux through glycolysis. The yeast senses glucose through the cell surface glucose sensors Rgt2 and Snf3, which serve as glucose receptors that generate the signal for induction of genes involved in glucose uptake and metabolism. Rgt2 and Snf3 detect high and low glucose concentrations, respectively, perhaps because of their different affinities for glucose. Here, we provide evidence that cell surface levels of glucose sensors are regulated by ubiquitination and degradation. The glucose sensors are removed from the plasma membrane through endocytosis and targeted to the vacuole for degradation upon glucose depletion. The turnover of the glucose sensors is inhibited in endocytosis defective mutants, and the sensor proteins with a mutation at their putative ubiquitin-acceptor lysine residues are resistant to degradation. Of note, the low affinity glucose sensor Rgt2 remains stable only in high glucose grown cells, and the high affinity glucose sensor Snf3 is stable only in cells grown in low glucose. In addition, constitutively active, signaling forms of glucose sensors do not undergo endocytosis, whereas signaling defective sensors are constitutively targeted for degradation, suggesting that the stability of the glucose sensors may be associated with their ability to sense glucose. Therefore, our findings demonstrate that the amount of glucose available dictates the cell surface levels of the glucose sensors and that the regulation of glucose sensors by glucose concentration may enable yeast cells to maintain glucose sensing activity at the cell surface over a wide range of glucose concentrations.

Cloning and characterization of heterologous transporters in Saccharomyces cerevisiae and identification of important amino acids for xylose utilization.

Efficient and specific transporters may enhance pentose uptake and metabolism by Saccharomyces cerevisiae. Eight heterologous sugar transporters were characterized in S. cerevisiae. The transporter Mgt05196p from Meyerozyma guilliermondii showed the highest xylose transport activity among them. Several key amino acid residues of Mgt05196p were suggested by structural and sequence analysis and characterized by site-directed mutagenesis. A conserved aromatic residue-rich motif (YFFYY, position 332-336) in the seventh trans-membrane span plays an important role in D-xylose transport activity. The phenyl ring of the residue at position 336 may take the function to prevent D-xylose from escaping during uptake. F432A and N360S mutations enhanced the D-xylose transport activities of Mgt05196p. Furthermore, mutant N360F specifically transported D-xylose without any glucose-inhibition, high lighting its potential application in constructing glucose-xylose co-fermentation strains for biomass refining.

Improvement of glucose uptake rate and production of target chemicals by overexpressing hexose transporters and transcriptional activator Gcr1 in Saccharomyces cerevisiae.

Metabolic engineering to increase the glucose uptake rate might be beneficial to improve microbial production of various fuels and chemicals. In this study, we enhanced the glucose uptake rate in Saccharomyces cerevisiae by overexpressing hexose transporters (HXTs). Among the 5 tested HXTs (Hxt1, Hxt2, Hxt3, Hxt4, and Hxt7), overexpression of high-affinity transporter Hxt7 was the most effective in increasing the glucose uptake rate, followed by moderate-affinity transporters Hxt2 and Hxt4. Deletion of STD1 and MTH1, encoding corepressors of HXT genes, exerted differential effects on the glucose uptake rate, depending on the culture conditions. In addition, improved cell growth and glucose uptake rates could be achieved by overexpression of GCR1, which led to increased transcription levels of HXT1 and ribosomal protein genes. All genetic modifications enhancing the glucose uptake rate also increased the ethanol production rate in wild-type S. cerevisiae. Furthermore, the growth-promoting effect of GCR1 overexpression was successfully applied to lactic acid production in an engineered lactic acid-producing strain, resulting in a significant improvement of productivity and titers of lactic acid production under acidic fermentation conditions.

Proton-associated sucrose transport of mammalian solute carrier family 45: an analysis in Saccharomyces cerevisiae.

The members of the solute carrier 45 (SLC45) family have been implicated in the regulation of glucose homoeostasis in the brain (SLC45A1), with skin and hair pigmentation (SLC45A2), and with prostate cancer and myelination (SLC45A3). However, apart from SLC45A1, a proton-associated glucose transporter, the function of these proteins is still largely unknown, although sequence similarities to plant sucrose transporters mark them as a putative sucrose transporter family. Heterologous expression of the three members SLC45A2, SLC45A3 and SLC45A4 in Saccharomyces cerevisiae confirmed that they are indeed sucrose transporters. [(14)C]Sucrose-uptake measurements revealed intermediate transport affinities with Km values of approximately 5 mM. Transport activities were best under slightly acidic conditions and were inhibited by the protonophore carbonyl cyanide m-chlorophenylhydrazone, demonstrating an H(+)-coupled transport mechanism. Na(+), on the other hand, had no effect on sucrose transport. Competitive inhibition assays indicated a possible transport also of glucose and fructose. Real-time PCR of mouse tissues confirmed mRNA expression of SLC45A2 in eyes and skin and of SLC45A3 primarily in the prostate, but also in other tissues, whereas SLC45A4 showed a predominantly ubiquitous expression. Altogether the results provide new insights into the physiological significance of SLC45 family members and challenge existing concepts of mammalian sugar transport, as they (i) transport a disaccharide, and (ii) perform secondary active transport in a proton-dependent manner.

Time-based comparative transcriptomics in engineered xylose-utilizing Saccharomyces cerevisiae identifies temperature-responsive genes during ethanol production.

Agricultural residues comprising lignocellulosic materials are excellent sources of pentose sugar, which can be converted to ethanol as fuel. Ethanol production via consolidated bioprocessing requires a suitable microorganism to withstand the harsh fermentation environment of high temperature, high ethanol concentration, and exposure to inhibitors. We genetically enhanced an industrial Saccharomyces cerevisiae strain, sun049, enabling it to uptake xylose as the sole carbon source at high fermentation temperature. This strain was able to produce 13.9 g/l ethanol from 50 g/l xylose at 38 °C. To better understand the xylose consumption ability during long-term, high-temperature conditions, we compared by transcriptomics two fermentation conditions: high temperature (38 °C) and control temperature (30 °C) during the first 12 h of fermentation. This is the first long-term, time-based transcriptomics approach, and it allowed us to discover the role of heat-responsive genes when xylose is the sole carbon source. The results suggest that genes related to amino acid, cell wall, and ribosomal protein synthesis are down-regulated under heat stress. To allow cell stability and continuous xylose uptake in order to produce ethanol, hexose transporter HXT5, heat shock proteins, ubiquitin proteins, and proteolysis were all induced at high temperature. We also speculate that the strong relationship between high temperature and increased xylitol accumulation represents the cell's mechanism to protect itself from heat degradation.

Effect of heterologous xylose transporter expression in Candida tropicalis on xylitol production rate.

Xylose utilization is inhibited by glucose uptake in xylose-assimilating yeasts, including Candida tropicalis, resulting in limitation of xylose uptake during the fermentation of glucose/xylose mixtures. In this study, a heterologous xylose transporter gene (At5g17010) from Arabidopsis thaliana was selected because of its high affinity for xylose and was codon-optimized for functional expression in C. tropicalis. The codon-optimized gene was placed under the control of the GAPDH promoter and was integrated into the genome of C. tropicalis strain LXU1 which is xyl2-disrupted and NXRG (codon-optimized Neurospora crassa xylose reductase) introduced. The xylose uptake rate was increased by 37-73 % in the transporter expression-enhanced strains depending on the glucose/xylose mixture ratio. The recombinant strain LXT2 in 500-mL flask culture using glucose/xylose mixtures showed a xylose uptake rate that was 29 % higher and a xylitol volumetric productivity (1.14 g/L/h) that was 25 % higher than the corresponding rates for control strain LXU1. Membrane protein extraction and Western blot analysis confirmed the successful heterologous expression and membrane localization of the xylose transporter in C. tropicalis.

Hexose transporters of a hemibiotrophic plant pathogen: functional variations and regulatory differences at different stages of infection.

Plant pathogenic fungi use a wide range of different strategies to gain access to the carbon sources of their host plants. The hemibiotrophic maize pathogen Colletotrichum graminicola (teleomorph Glomerella graminicola) colonizes its host plants, and, after a short biotrophic phase, switches to destructive, necrotrophic development. Here we present the identification of five hexose transporter genes from C. graminicola, CgHXT1 to CgHXT5, the functional characterization of the encoded proteins, and detailed expression studies for these genes during vegetative and pathogenic development. Whereas CgHXT4 is expressed under all conditions analyzed, transcript abundances of CgHXT1 and CgHXT3 are transiently up-regulated during the biotrophic phase, and CgHXT2 and CgHXT5 are expressed exclusively during necrotrophic development. Analyses of the encoded proteins characterized CgHXT5 as a low-affinity/high-capacity hexose transporter with a narrow substrate specificity for glucose and mannose. In contrast, CgHXT1 to CgHXT3 are high affinity/low capacity transporters that also accept other substrates, including fructose, galactose, or xylose. CgHXT4, the largest of the identified proteins, has only little transport activity and may function as a sugar sensor. Phylogenetic studies revealed hexose transporters closely related to the five CgHXT proteins also in other pathogenic fungi suggesting conserved functions of these proteins during fungal pathogenesis.

Metabolic engineering of D-xylose pathway in Clostridium beijerinckii to optimize solvent production from xylose mother liquid.

Clostridium beijerinckii is an attractive butanol-producing microbe for its advantage in co-fermenting hexose and pentose sugars. However, this Clostridium strain exhibits undesired efficiency in utilizing D-xylose, one of the major building blocks contained in lignocellulosic materials. Here, we reported a useful metabolic engineering strategy to improve D-xylose consumption by C. beijerinckii. Gene cbei2385, encoding a putative D-xylose repressor XylR, was first disrupted in the C. beijerinckii NCIMB 8052, resulting in a significant increase in D-xylose consumption. A D-xylose proton-symporter (encoded by gene cbei0109) was identified and then overexpressed to further optimize D-xylose utilization, yielding an engineered strain 8052xylR-xylT(ptb) (xylR inactivation plus xylT overexpression driven by ptb promoter). We investigated the strain 8052xylR-xylT(ptb) in fermenting xylose mother liquid, an abundant by-product from industrial-scale xylose preparation from corncob and rich in D-xylose, finally achieving a 35% higher Acetone, Butanol and Ethanol (ABE) solvent titer (16.91 g/L) and a 38% higher yield (0.29 g/g) over those of the wild-type strain. The strategy used in this study enables C. beijerinckii more suitable for butanol production from lignocellulosic materials.

Functional analysis of an Arabidopsis thaliana abiotic stress-inducible facilitated diffusion transporter for monosaccharides.

Sugars play indispensable roles in biological reactions and are distributed into various tissues or organelles via transporters in plants. Under abiotic stress conditions, plants accumulate sugars as a means to increase stress tolerance. Here, we report an abiotic stress-inducible transporter for monosaccharides from Arabidopsis thaliana that is termed ESL1 (ERD six-like 1). Expression of ESL1 was induced under drought and high salinity conditions and with exogenous application of abscisic acid. Promoter analyses using beta-glucuronidase and green fluorescent protein reporters revealed that ESL1 is mainly expressed in pericycle and xylem parenchyma cells. The fluorescence of ESL1-green fluorescent protein-fused protein was detected at tonoplast in transgenic Arabidopsis plants and tobacco BY-2 cells. Furthermore, alanine-scanning mutagenesis revealed that an N-terminal LXXXLL motif in ESL1 was essential for its localization at the tonoplast. Transgenic BY-2 cells expressing mutated ESL1, which was localized at the plasma membrane, showed an uptake ability for monosaccharides. Moreover, the value of K(m) for glucose uptake activity of mutated ESL1 in the transgenic BY-2 cells was extraordinarily high, and the transport activity was independent from a proton gradient. These results indicate that ESL1 is a low affinity facilitated diffusion transporter. Finally, we detected that vacuolar invertase activity was increased under abiotic stress conditions, and the expression patterns of vacuolar invertase genes were similar to that of ESL1. Under abiotic stress conditions, ESL1 might function coordinately with the vacuolar invertase to regulate osmotic pressure by affecting the accumulation of sugar in plant cells.

Engineering topology and kinetics of sucrose metabolism in Saccharomyces cerevisiae for improved ethanol yield.

Sucrose is a major carbon source for industrial bioethanol production by Saccharomyces cerevisiae. In yeasts, two modes of sucrose metabolism occur: (i) extracellular hydrolysis by invertase, followed by uptake and metabolism of glucose and fructose, and (ii) uptake via sucrose-proton symport followed by intracellular hydrolysis and metabolism. Although alternative start codons in the SUC2 gene enable synthesis of extracellular and intracellular invertase isoforms, sucrose hydrolysis in S. cerevisiae predominantly occurs extracellularly. In anaerobic cultures, intracellular hydrolysis theoretically enables a 9% higher ethanol yield than extracellular hydrolysis, due to energy costs of sucrose-proton symport. This prediction was tested by engineering the promoter and 5' coding sequences of SUC2, resulting in predominant (94%) cytosolic localization of invertase. In anaerobic sucrose-limited chemostats, this iSUC2-strain showed an only 4% increased ethanol yield and high residual sucrose concentrations indicated suboptimal sucrose-transport kinetics. To improve sucrose-uptake affinity, it was subjected to 90 generations of laboratory evolution in anaerobic, sucrose-limited chemostat cultivation, resulting in a 20-fold decrease of residual sucrose concentrations and a 10-fold increase of the sucrose-transport capacity. A single-cell isolate showed an 11% higher ethanol yield on sucrose in chemostat cultures than an isogenic SUC2 reference strain, while transcriptome analysis revealed elevated expression of AGT1, encoding a disaccharide-proton symporter, and other maltose-related genes. After deletion of both copies of the duplicated AGT1, growth characteristics reverted to that of the unevolved SUC2 and iSUC2 strains. This study demonstrates that engineering the topology of sucrose metabolism is an attractive strategy to improve ethanol yields in industrial processes.

Stepwise increase of resveratrol biosynthesis in yeast Saccharomyces cerevisiae by metabolic engineering.

Resveratrol is a unique, natural polyphenolic compound with diverse health benefits. In the present study, we attempted to improve resveratrol biosynthesis in yeast by different methods of metabolic engineering. We first mutated and then re-synthesized tyrosine ammonia lyase (TAL) by replacing the bacteria codons with yeast-preferred codons, which increased translation and improved p-coumaric acid and resveratrol biosynthesis drastically. We then demonstrated that low-affinity, high-capacity bacterial araE transporter could enhance resveratrol accumulation, without transporting resveratrol directly. Yeast cells carrying the araE gene produced up to 2.44-fold higher resveratrol than control cells. For commercial applications, resveratrol biosynthesis was detected in sucrose medium and fresh grape juice using our engineered yeast cells. In collaboration with the Chaumette Winery of Missouri, we were able to produce resveratrol-containing white wines, with levels comparable to the resveratrol levels found in most red wines.

Kinetic modelling reveals current limitations in the production of ethanol from xylose by recombinant Saccharomyces cerevisiae.

Saccharomyces cerevisiae lacks the ability to ferment the pentose sugar xylose that is the second most abundant sugar in nature. Therefore two different xylose catabolic pathways have been heterologously expressed in S. cerevisiae. Whereas the xylose reductase (XR)-xylitol dehydrogenase (XDH) pathway leads to the production of the by-product xylitol, the xylose isomerase (XI) pathway results in significantly lower xylose consumption. In this study, kinetic models including the reactions ranging from xylose transport into the cell to the phosphorylation of xylulose to xylulose 5-P were constructed. They were used as prediction tools for the identification of putative targets for the improvement of xylose utilization in S. cerevisiae strains engineered for higher level of the non-oxidative pentose phosphate pathway (PPP) enzymes, higher xylulokinase and inactivated GRE3 gene encoding an endogenous NADPH-dependent aldose reductase. For both pathways, the in silico analyses identified a need for even higher xylulokinase (XK) activity. In a XR-XDH strain expressing an integrated copy of the Escherichia coli XK encoding gene xylB about a six-fold reduction of xylitol formation was confirmed under anaerobic conditions. Similarly overexpression of the xylB gene in a XI strain increased the aerobic growth rate on xylose by 21%. In contrast to the in silico predictions, the aerobic growth also increased 24% when the xylose transporter gene GXF1 from Candida intermedia was overexpressed together with xylB in the XI strain. Under anaerobic conditions, the XI strains overexpressing xylB gene and the combination of xylB and GFX1 genes consumed 27% and 37% more xylose than the control strain.

Quantitative analysis of energy transfer between fluorescent proteins in CFP-GBP-YFP and its response to Ca2+.

This article reports the full characterisation of the optical properties of a biosynthesised protein consisting of fused cyan fluorescent protein, glucose binding protein and yellow fluorescent protein. The cyan and yellow fluorescent proteins act as donors and acceptors for intramolecular fluorescence resonance energy transfer. Absorption, fluorescence, excitation and fluorescence decays of the compound protein were measured and compared with those of free fluorescent proteins. Signatures of energy transfer were identified in the spectral intensities and fluorescence decays. A model describing the fluorescence properties including energy transfer in terms of rate equations is presented and all relevant parameters are extracted from the measurements. The compound protein changes conformation on binding with calcium ions. This is reflected in a change of energy transfer efficiency between the fluorescent proteins. We track the conformational change and the kinetics of the calcium binding reaction from fluorescence intensity and decay measurements and interpret the results in light of the rate equation model. This visualisation of change in protein conformation has the potential to serve as an analytical tool in the study of protein structure changes in real time, in the development of biosensor proteins and in characterizing protein-drug interactions.

Asymmetric signal transduction through paralogs that comprise a genetic switch for sugar sensing in Saccharomyces cerevisiae.

Efficient uptake of glucose is especially critical to Saccharomyces cerevisiae because its preference to ferment this carbon source demands high flux through glycolysis. Glucose induces expression of HXT genes encoding hexose transporters through a signal generated by the Snf3 and Rgt2 glucose sensors that leads to depletion of the transcriptional regulators Mth1 and Std1. These paralogous proteins bind to Rgt1 and enable it to repress expression of HXT genes. Here we show that Mth1 and Std1 can substitute for one another and provide nearly normal regulation of their targets. However, their roles in the glucose signal transduction cascade have diverged significantly. Mth1 is the prominent effector of Rgt1 function because it is the more abundant of the two paralogs under conditions in which both are active (in the absence of glucose). Moreover, the cellular level of Mth1 is quite sensitive to the amount of available glucose. The abundance of Std1 protein, on the other hand, remains essentially constant over a similar range of glucose concentrations. The signal generated by low levels of glucose is amplified by rapid depletion of Mth1; the velocity of this depletion is dependent on both its rate of degradation and swift repression of MTH1 transcription by the Snf1-Mig1 glucose repression pathway. Quantitation of the contributions of Mth1 and Std1 to regulation of HXT expression reveals the unique roles played by each paralog in integrating nutrient availability with metabolic capacity: Mth1 is the primary regulator; Std1 serves to buffer the response to glucose.

Mangiferin promotes intestinal elimination of uric acid by modulating intestinal transporters.

Increasing evidence shows that the intestinal tract plays an important role in maintaining urate homeostasis and might be a potential therapeutic target for hyperuricaemia. However, uric acid-lowering drugs available in the clinic do not target intestinal excretion as a therapeutic strategy. We previously reported that mangiferin had potent hypouricaemic effects in hyperuricaemic animals. However, the underlying mechanisms are not completely clear. Here, we investigated the effects of mangiferin on the intestinal excretion of urate and its underlying mechanisms. The data revealed that mangiferin concentration-dependently promoted the intestinal secretion of endogenous urate in in situ intestinal closed loops in normal and hyperuricaemic mice, as well as inhibited the absorption of exogenous uric acid perfused into the intestinal loops in rats. Administration of mangiferin not only decreased the serum urate levels in the hyperuricaemic mice but also increased the protein expression of ATP-binding cassette transporter, subfamily G, member 2 (ABCG2) and inhibited the protein expression of glucose transporter 9 (GLUT 9) in the intestine. These findings suggested that intestinal ABCG2 and GLUT9 might be pivotal and possible action sites for the observed hypouricaemic effects. Moreover, no significant changes in intestinal xanthine oxidoreductase activities were observed, suggesting that mangiferin did not affect intestinal uric acid generation in the hyperuricaemic mice. Overall, promoting intestinal elimination of urate by upregulating ABCG2 expression and downregulating GLUT9 expression might be an important mechanism underlying mangiferin lowering serum uric acid levels. Mangiferin supplementation might be beneficial for the prevention and treatment of hyperuricaemia.

Expression and compartmentalization of caveolin in adipose cells: coordinate regulation with and structural segregation from GLUT4.

Native rat adipocytes and the mouse adipocyte cell line, 3T3-L1, possess transport vesicles of apparently uniform composition and size which translocate the tissue-specific glucose transporter isoform, GLUT4, from an intracellular pool to the cell surface in an insulin-sensitive fashion. Caveolin, the presumed structural protein of caveolae, has also been proposed to function in vesicular transport. Thus, we studied the expression and subcellular distribution of caveolin in adipocytes. We found that rat fat cells express the highest level of caveolin protein of any tissue studied, and caveolin is also expressed at high levels in cardiac muscle, another tissue possessing insulin responsive GLUT4 translocation. Both proteins are absent from 3T3-L1 fibroblasts and undergo a dramatic coordinate increase in expression upon differentiation of these cells into adipocytes. However, unlike GLUT4 in rat adipocytes not exposed to insulin, the majority of caveolin is present in the plasma membrane. In native rat adipocytes, intracellular GLUT4 and caveolin reside in vesicles practically indistinguishable by their size and buoyant density in sucrose gradients, and both proteins show insulin-dependent translocation to the cell surface. However, by immunoadsorption of GLUT4-containing vesicles with anti-GLUT4 antibody, we show that these vesicles have no detectable caveolin, and therefore, this protein is present in a distinct vesicle population. Thus, caveolin has no direct structural relation to the organization of the intracellular glucose transporting machinery in fat cells.

A double leucine within the GLUT4 glucose transporter COOH-terminal domain functions as an endocytosis signal.

The unique COOH-terminal 30-amino acid region of the adipocyte/skeletal muscle glucose transporter (GLUT4) appears to be a major structural determinant of this protein's perinuclear localization, from where it is redistributed to the cell surface in response to insulin. To test whether an underlying mechanism of this domain's function involves glucose transporter endocytosis rates, transfected cells were generated expressing exofacial hemagglutinin epitope (HA)-tagged erythrocyte/brain glucose transporter (GLUT1) or a chimera containing the COOH-terminal 30 amino acids of GLUT4 substituted onto this GLUT1 construct. Incubation of COS-7 or CHO cells expressing the HA-tagged chimera with anti-HA antibody at 37 degrees resulted in an increased rate of antibody internalization compared to cells expressing similar levels of HA-tagged GLUT1, which displays a cell surface disposition. Colocalization of the internalized anti-HA antibody in vesicular structures with internalized transferrin and with total transporters was established by digital imaging microscopy, suggesting the total cellular pool of transporters are continuously recycling through the coated pit endocytosis pathway. Mutation of the unique double leucines 489 and 490 in the rat GLUT4 COOH-terminal domain to alanines caused the HA-tagged chimera to revert to the slow endocytosis rate and steady-state cell surface display characteristic of GLUT1. These results support the hypothesis that the double leucine motif in the GLUT4 COOH terminus operates as a rapid endocytosis and retention signal in the GLUT4 transporter, causing its localization to intracellular compartments in the absence of insulin.