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PubMed1991

Normalization of blood glucose in diabetic rats with phlorizin treatment reverses insulin-resistant glucose transport in adipose cells without restoring glucose transporter gene expression.

Evidence is emerging for a direct role of glucose, independent of changes in insulin, in the regulation of cellular glucose transport and glucose utilization in vivo. In this study we investigate potential cellular and molecular mechanisms for this regulatory effect of glucose by determining how normalization of glycemia without insulin therapy in diabetic rats influences 3-O-methylglucose transport and the expression and translocation of two genetically distinct species of glucose transporters (GTs) in adipose cells. These results are compared with alterations in glucose disposal in vivo measured by euglycemic clamp. In rats rendered diabetic by 90% pancreatectomy, insulin-stimulated glucose transport in adipose cells is decreased 50% in parallel with reduced insulin-mediated glucose disposal in vivo. Levels of adipose/muscle GTs measured by immunoblotting are decreased in adipose cell subcellular membrane fractions, as are the corresponding mRNA levels assessed by Northern blotting of total adipose cell RNA. Normalization of blood glucose in diabetic rats with phlorizin, which impairs renal tubular glucose reabsorption and thus enhances glucose excretion, restores insulin-stimulated glucose transport in adipose cells and insulin-mediated glucose disposal in vivo. Importantly, levels of the adipose/muscle GT protein remain 43% reduced in the low-density microsomes in the basal state and 46% reduced in the plasma membranes in the insulin-stimulated state. Adipose/muscle GT mRNA levels remain approximately 50% depressed. Levels of the HepG2/brain GT protein and mRNA are unaltered by diabetes or phlorizin treatment. Thus, changes in ambient glucose independent of changes in ambient insulin can regulate the glucose transport response to insulin in isolated adipose cells and changes in responsiveness parallel alterations in glucose uptake in vivo. Since this effect can occur without alteration in the expression of the two species of glucose transporters present in adipose cells or in their translocation to the plasma membrane in response to insulin, it may result from changes in GT functional activity.

Adipose Tissue↗
PubMed1979

Effects of insulin on glucose metabolism and glucose transport in fat cells of hormone-treated hypophysectomized rats: evidence that growth hormone restricts glucose transport.

In earlier studies we have shown that insulin does not stimulate glucose incorporation in adipocytes of hypophysectomized (hypox) rats. Basal glucose incorporation is decreased, although basal 3-O-methylglycose transport is very rapid and cannot be further stimulated by insulin. In this study we treated hypox rats with human GH, ACTH, and T3, alone or in combination, and examined the effects of insulin on glucose incorporation into fat cells and on 3-O-methylglucose transport. The results show that chronic administration of T3 alone to hypox rats partially restores glucose incorporation into fat cells and, in combination with ACTH, completely restores this incorporation. The two hormones have no effect on the glucose carrier system. The transport rate under T3 and ACTH replacement therapy continues to proceed at a maximal rate, so that basal glucose incorporation is high but not further enhanced by insulin. In contrast, administration of human GH to hypox rats does not influence glucose incorporation but has a marked effect on glucose transport. The basal glucose transport rate returns toward normal and again responds to insulin. This suggests 1) that enzyme activities responsible for the lipogenetic capacity of the fat cell are decreased in hypox rats and returned toward normal by the combined T3/ACTH treatment, and 2) that the limitation of glucose transport in the fat cell is controlled by GH. GH seems to induce a change of the glucose-carrier system; it leads to a restriction of glucose transport, which is acutely modulated by insulin.

Adipose Tissue↗
PubMed1987

Stereospecificity of triiodothyronine transport into brain, liver, and salivary gland: role of carrier- and plasma protein-mediated transport.

The stereospecificity of T3 transport through the walls of the brain capillary, i.e. the blood-brain barrier (BBB), and the salivary gland capillary and through the hepatocyte plasma membrane was studied with a tissue-sampling single injection technique in rats. In the absence of plasma proteins, the ED50 of inhibition of the saturable transport of [125I]L-T3 through the BBB was 1 microM for unlabeled L-T3 and 9 microM for unlabeled D-T3. The brain extraction of [125I]D-T3, 5.9 +/- 0.1% (+/- SE), was about one third that of [125I]L-T3. Conversely, no saturable and no stereospecific T3 transport was observed for the salivary gland capillary, which, unlike the brain capillary, is porous. The hepatic extraction of T3 was minimally stereospecific in the absence of plasma proteins. In the presence of 5 g/dl bovine albumin, the fraction of circulating D- or L-T3 that was available for transport into liver (50-100%) was many-fold greater than the fraction that was free in vitro (approximately 2%); therefore, both D-T3 and L-T3 were available for uptake by liver from the circulating albumin-bound pool. This plasma protein-mediated transport of T3 is believed to represent a process of enhanced dissociation of T3 from the albumin-binding site, since the plasma protein per se is not significantly taken up by liver on a single pass. However, in the presence of 5 g/dl bovine albumin, the extravascular hepatic extraction of [125I]D-T3 (50 +/- 2%) was nearly half that for [125I]T3 (93 +/- 12%), although no significant difference in the in vitro binding of [125I]D-T3 and [125I]L-T3 to 5 g/dl bovine albumin was found with equilibrium dialysis. In addition, the isoelectric point of bovine albumin bound to [125I] L-T3 (5.1) was higher than that of bovine albumin bound to [125I] D-T3 (5.0), as determined by isoelectric focusing, which indicates that the surface of the bovine albumin molecule is slightly more positive when the protein binds L-T3 as opposed to D-T3. The isoelectric focusing and in vivo transport data together suggest that the interaction between the surfaces of the plasma protein and the hepatic microcirculation that is presumed to cause enhanced hormone dissociation from the protein-binding site is electrostatic in nature. These studies (1) show that the BBB thyroid hormone transport system is sharply stereospecific, and this property is probably a major factor underlying the low biological potency of D-T3 in brain.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1994

Relationship between thyroid hormone transport and neutral amino acid transport in JAR human choriocarcinoma cells.

The relationship between the transport of the thyroid hormone T3 and the transport of neutral amino acids was investigated in JAR human placental choriocarcinoma cells. The uptake of leucine, mediated by the amino acid transport system L, was inhibited by T3 and T4, and the nature of inhibition was competitive. Uptake of T3 into the cells was predominantly Na+ independent, and so was that of leucine. However, although an acidic extracellular pH stimulated leucine uptake, the uptake of T3 remained unaffected. In addition, leucine failed to inhibit T3 uptake. The aromatic amino acids phenylalanine and tryptophan were found to inhibit the uptake of T3, but these two amino acids were transported into the cells predominantly via system L. The amino acid transport system T, which is specific for aromatic amino acids, was not detectable in these cells. Treatment of the cells with the calmodulin antagonist CGS 9343 B stimulated the uptake of leucine and tryptophan, but inhibited the uptake of T3. Kinetic analysis of T3 uptake revealed the presence of a single saturable system for this hormone in these cells, and the Michaelis-Menten constant for this system was 0.77 +/- 0.06 microM. Metabolic poisons that interfere with the cellular generation of ATP had no effect on the uptake of T3. It is concluded that in placental choriocarcinoma cells, 1) T3 and T4 are high affinity competitive inhibitors of the amino acid transport system L, 2) uptake of T3 occurs via a specific Na(+)-independent, energy-independent, and saturable mechanism that is unrelated to the amino acid transport systems L and T, 3) the aromatic amino acids phenylalanine and tryptophan interact, although weakly, with the T3 uptake system, and 4) calmodulin-dependent processes participate in the regulation of the T3 uptake system.

Amino Acids↗
PubMed1996

Effect of 3,5,3'-triiodothyronine on maturation of rat renal phosphate transport: kinetic characteristics and phosphate transporter messenger ribonucleic acid and protein abundance.

The plasma concentration of T3 increases during the third postnatal week in rats when the young are weaned. Renal phosphate (Pi) transport matures during this period, with increases in the carrier affinity for Na-cotransported Pi(1/K(m)) and in the maximal transport rate (Vmax). As prevention of weaning prevents the normal increase in the plasma T3 concentration and blocks the maturation of Pi transport, this study examines the influence of T3 on Pi transport maturation. The brush border membranes from 21-day-old rats rendered hypothyroid with 6-n-propyl-2-thiouracil (PTU) or prevented from weaning (PW) were examined. These two treatments effectively prevented normal maturation, as the apparent K(m) was significantly higher in the PTU (0.243 +/- 0.04) and PW (0.159 +/- 0.008) rats than in controls [0.111 +/- 0.032 (n = 6; P < 0.05) and 0.104 +/- 0.005 (n = 6; P < 0.05)], and the Vmax was significantly lower [3192 +/- 170 vs. 4269 +/- 343 (n = 6; P < 0.01) and 3479 +/- 478 vs. 4184 +/- 368 (n = 6; P < 0.05)]. Administration of T3 to PTU and PW rats restored the apparent K(m) and V(max) to control values. The amounts of Na-Pi co-transporter messenger RNA and protein were also determined. The amounts of NaPi-2 messenger RNA and protein were decreased about 5-fold in PTU and PW rats, and T3 restored them to control levels. The effect of T3 on the maturation of Pi transport was independent of PTH, GH, or glucocorticoids. Thus, thyroid hormone helps control the maturation of Pi transport in the rat during weaning, and thyroid hormone may be a dominant triggering factor in this process.

Aging↗
PubMed2002

Identification of a novel human organic anion transporting polypeptide as a high affinity thyroxine transporter.

Transport of various amphipathic organic compounds is mediated by organic anion transporting polypeptides (OATPs in humans, Oatps in rodents), which belong to the solute carrier family 21A (SLC21A/Slc21a). Several of these transporters exhibit a broad and overlapping substrate specificity and are expressed in a variety of different tissues. We have isolated and functionally characterized OATP-F (SLC21A14), a novel member of the OATP family. The cDNA (3059 bp) contains an open reading frame of 2136 bp encoding a protein of 712 amino acids. Its gene containing 15 exons is located on chromosome 12p12. OATP-F exhibits 47-48% amino acid identity with OATP-A, OATP-C, and OATP8, the genes of which are clustered on chromosome 12p12. OATP-F is predominantly expressed in multiple brain regions and Leydig cells of the testis. OATP-F mediates high affinity transport of T(4) and reverse T(3) with apparent K(m) values of approximately 90 nM and 128 nM, respectively. Substrates less well transported by OATP-F include T(3), bromosulfophthalein, estrone-3-sulfate, and estradiol-17beta-glucuronide. Furthermore, OATP-F-mediated T(4) uptake could be cis-inhibited by L-T(4) and D-T(4), but not by 3,5-diiodothyronine, indicating that T(4) transport is not stereospecific, but that 3',5'-iodination is important for efficient transport by OATP-F. Thus, in contrast to most other family members, OATP-F has a more selective substrate preference and may play an important role in the disposition of thyroid hormones in brain and testis.

Amino Acid Sequence↗
PubMed1994

A new family of proteins (rBAT and 4F2hc) involved in cationic and zwitterionic amino acid transport: a tale of two proteins in search of a transport function.

The currently identified cDNA clones of mammalian amino acid transporters can be grouped into five different families. One family is composed of the proteins rBAT and the heavy chain (hc) of the cell surface antigen 4F2. RNAs encoding these two proteins induce a system b(o,+)-like (rBAT) and a system y+L-like (4F2hc) activity in Xenopus oocytes. Surprisingly, rBAT and 4F2hc do not seem to be pore-forming proteins. This finding supports the hypothesis that rBAT and 4F2hc are subunits or modulators of the corresponding amino acid transport systems. Expression of rBAT in oocytes induces high-affinity transport of cystine, which is shared with transport of cationic and zwitterionic amino acids. The rBAT gene is expressed mainly in kidney and small intestine. The rBAT protein is localized to the microvilli of proximal straight tubules of the kidney and mucosa from the small intestine. This finding is consistent with the involvement of rBAT in a high-affinity resorption system for cystine in the proximal straight tubule of the nephron. All of these characteristics suggest that rBAT is a good candidate for a cystinuria gene. Cystinuria is an inheritable defect in high-affinity transport of cystine, shared with cationic amino acids, through epithelial cells of the renal tubule and intestinal tract. Very recently, point missense mutations have been found in the rBAT gene of cystinuria patients. The most frequent rBAT mutation, M467T (threonine substitution of methionine at residue 467) nearly abolished the amino acid transport activity elicited by rBAT in oocytes. This result offers convincing evidence that rBAT is a cystinuria gene. Biochemical, cytological and genetic approaches are now needed to delineate the mechanism of action of rBAT and 4F2hc in the transport of amino acids.

Amino Acid Transport Systems, Basic↗
PubMed1995

[Kinetic analysis of hepatobiliary transport of drugs: importance of carrier-mediated transport].

The liver is the major organ involved in the metabolism and elimination of xenobiotics. Evaluating accurately hepatic clearance is very important for predicting the pharmacological effect and/or side-effects of drugs, as well as changes in drug disposition during disease. Recently, for many endogenous and exogenous compounds (including drugs), it has been reported that carrier-mediated transport contributes to hepatic uptake and/or biliary excretion. In particular, primary active transport mechanisms have been shown to be responsible for the biliary excretion of anticancer drugs, endogenous bile acids and organic anions including glutathione and glucuronic acid conjugates. We have found that a rate-limiting step for several drugs in terms of the hepatic clearance was the membrane transport process. For these drugs, such a saturable transport process can be one of the major determinants which influence not only hepatic clearance itself but also disposition (in other words, plasma elimination) in the whole body. We reviewed the carrier-mediated transport mechanisms involved in the hepatic uptake and biliary excretion processes, the multiplicity of transport systems, and further, the physiological meaning of these complex transport systems in the body.

Animals↗
PubMed2002

Transepithelial transport of fluorescein in Caco-2 cell monolayers and use of such transport in in vitro evaluation of phenolic acid availability.

Fluorescein is a marker-dye customary applied to the evaluation of tight-junctional permeability of epithelial cell monolayers. However, the true mechanism for the permeation has not been elucidated. Transepithelial transport of fluorescein in Caco-2 cell monolayers was therefore examined. Fluorescein transport was dependent on pH, and in a vectorical way in the apical-basolateral direction, but it was independent of the tight-junctional permeability of monolayers of these human intestinal cells. The permeation of fluorescein was concentration-dependent and saturable; the Michaelis constant was 7.7 mM and the maximum velocity was 40.3 nmol min(-1) (mg protein)(-1). Benzoic acid competitively inhibited fluorescein transport, suggesting that fluorescein is transported by a monocarboxylic acid transporter (MCT). Antioxidative polyphenolic compounds such as ferulic acid from dietary sources, competitively inhibited the permeation of fluorescein. These compounds probably share a transport carrier with fluorescein. Measurement of the effects of phenolic acids on fluorescein transport across Caco-2 monolayers would be a useful way to evaluate the intestinal absorption or bioavailability of dietary phenolic acids.

Benzoic Acid↗
PubMed2006

Catalyst transport in corn stover internodes: elucidating transport mechanisms using Direct Blue-I.

The transport of catalysts (chemicals and enzymes) within plant biomass is believed to be a major bottleneck during thermochemical pretreatment and enzymatic conversion of lignocellulose. Subjecting biomass to size reduction and mechanical homogenization can reduce catalyst transport limitations; however, such processing adds complexity and cost to the overall process. Using high-resolution light microscopy, we have monitored the transport of an aqueous solution of Direct Blue-I (DB-I) dye through intact corn internodes under a variety of impregnation conditions. DB-I is a hydrophilic anionic dye with affinity for cellulose. This model system has enabled us to visualize likely barriers and mechanisms of catalyst transport in corn stems. Microscopic images were compared with calculated degrees of saturation (i.e., volume fraction of internode void space occupied by dye solution) to correlate impregnation strategies with dye distribution and transport mechanisms. Results show the waxy rind exterior and air trapped within individual cells to be the major barriers to dye transport, whereas the vascular bundles, apoplastic continuum (i.e., the intercellular void space at cell junctions), and fissures formed during the drying process provided the most utilized pathways for transport. Although representing only 20-30% of the internode volume, complete saturation of the apoplast and vascular bundles by fluid allowed dye contact with a majority of the cells in the internode interior.

Azo Compounds↗
PubMed2003

Fatty acids alter monolayer integrity, paracellular transport, and iron uptake and transport in Caco-2 cells.

The Caco-2 cell line was used as a model to determine if the type of fatty acid, unsaturated versus saturated, differentially altered the uptake and transport of iron in the human intestine and if the changes were the result of alterations in monolayer integrity and paracellular transport. Cells were cultured in either a lower-iron or normal-iron medium and incubated with a bovine serum albumin control, linoleate, oleate, palmatate, or stearate. Oleate, palmatate, and stearate enhanced (p < 0.05) iron uptake in cells grown in lower iron. The fatty acid effect on iron uptake by cells grown in normal iron was not as pronounced. Iron transport was not affected (p > 0.05) by an interaction between the type of medium (iron concentration) and the type of fatty acid. Iron transport was enhanced (p < 0.05) with palmatate and stearate. Various indicators of monolayer integrity and paracellular transport were also affected by the fatty acids, thus impacting iron uptake and transport. These results indicate that oleate, palmatate, and stearic can enhance iron uptake and transport; however, this enhancement may be the result of alterations in the integrity of the intestine.

Biological Transport↗
PubMed2004

Regulation of phosphate (Pi) transport and NaPi-III transporter (Pit-1) mRNA in rat osteoblasts.

In osteoblasts only the type III Na(+)-dependent phosphate (NaPi) transporter isoforms Pit-1 and Pit-2 have been identified. We tested the effects of extracellular Pi, Ca(2+) and IGF-I on Na(d)Pi transport and Pit-1 or Pit-2 mRNA expression in rat osteoblastic (PyMS) cells. The v(max) of Na(d)Pi transport was higher in cells kept in Pi-free, serum-free medium for 24 h than in controls at 1 mM Pi (2.47+/-0.20 vs 1.83+/-0.17 nmol/mg protein x 10 min). The apparent affinity constant (K(M)) for Pi remained unchanged. Pi withdrawal for 24 h did not impair cell viability whereas increasing the extracellular Pi to 5 mM resulted in cell death. Pit-1 (but not Pit-2) mRNA was upregulated following Pi deprivation, Ca(2+) treatment or after treatment with 1 nM IGF-I, known to stimulate Na(d)Pi transport and cell proliferation. IGF-I also stimulated Na(d)Pi transport and Pit-1 mRNA in primary rat calvarial osteoblasts. Expression of Pit-1 mRNA in vivo and the coordinate regulation of Pit-1 mRNA and Pi transport in osteoblastic cells suggest that Pit-1 is a candidate transporter of physiological relevance in bone.

Alkaline Phosphatase↗
PubMed2001

Contribution of specific transport systems to anthracycline transport in tumor and normal cells.

Anthracycline antibiotics are very effective neoplastic agents widely used clinically. However, because of their many adverse effects (e.g. cardiotoxicity, leukopenia and alopecia), their clinical use has been limited. In order to minimize their adverse effects in clinical cancer chemotherapy, anthracyclines must be selectively transported into tumor cells. If there are differences in transport characteristics between tumor and normal cells, it should be possible to establish a strategy for selectively delivering anthracyclines to tumor cells on the basis of the differences. In human cultured leukemia HL60 cells, as tumor cells, and human fresh mononuclear cells, as normal cells, doxorubicin, pirarubicin, daunorubicin and idarubicin were incorporated via a common carrier-mediated system, but the carriers were different in the two cell types. In HL60 cells, it was indicated that a nucleoside transport system contributed, at least in part, to the transport of doxorubicin and pirarubicin, but not daunorubicin and idarubicin, and its contribution to pirarubicin transport was found in other tumor cells, i.e. mouse ovarian sarcoma M5076 and Ehrlich ascites carcinoma cells. On the other hand, in mononuclear cells, there was no involvement of a nucleoside transport system for the four anthracyclines examined. Therefore, we thought that with the modification of an anthracycline molecule as a substrate for the nucleoside transport system, the anthracycline could be delivered selectively to tumor cells.

Animals↗
PubMed2004

The testing and modification of a commercially available transport medium for the transportation of pure cultures of Haemophilus paragallinarum for serotyping.

Haemophilus paragallinarum, the causative agent of infectious coryza in poultry, is an extremely fastidious organism requiring specific growth conditions for isolation. For complete control of the disease in regions where more that one of the serovars of the different serogroups occurs, it is essential that the bacterium causing the problem be isolated and serotyped. This work describes the modification and testing of transport media, which will ensure the survival of the causative agents in suspected infectious coryza cases for transport to a laboratory where the bacterium can be isolated and serotyped. The various transport media used are based on commercially available Amies Transport Medium supplemented with the different supplements used for the growth of H. paragallinarum. It was established that the bacterium remains viable for up to 18 days in Amies Transport Medium containing all the supplements when stored at 4 degrees C or 37 degrees C. At room temperature or 25 degrees C, there was no difference in the survival of H. paragallinarum in commercial Amies Transport Medium (without charcoal) and Amies Transport Medium with supplements.

Animals↗
PubMed2000

Neurofilaments are transported rapidly but intermittently in axons: implications for slow axonal transport.

Slow axonal transport conveys cytoskeletal proteins from cell body to axon tip. This transport provides the axon with the architectural elements that are required to generate and maintain its elongate shape and also generates forces within the axon that are necessary for axon growth and navigation. The mechanisms of cytoskeletal transport in axons are unknown. One hypothesis states that cytoskeletal proteins are transported within the axon as polymers. We tested this hypothesis by visualizing individual cytoskeletal polymers in living axons and determining whether they undergo vectorial movement. We focused on neurofilaments in axons of cultured sympathetic neurons because individual neurofilaments in these axons can be visualized by optical microscopy. Cultured sympathetic neurons were infected with recombinant adenovirus containing a construct encoding a fusion protein combining green fluorescent protein (GFP) with the heavy neurofilament protein subunit (NFH). The chimeric GFP-NFH coassembled with endogenous neurofilaments. Time lapse imaging revealed that individual GFP-NFH-labeled neurofilaments undergo vigorous vectorial transport in the axon in both anterograde and retrograde directions but with a strong anterograde bias. NF transport in both directions exhibited a broad spectrum of rates with averages of approximately 0.6-0.7 microm/sec. However, movement was intermittent, with individual neurofilaments pausing during their transit within the axon. Some NFs either moved or paused for the most of the time they were observed, whereas others were intermediate in behavior. On average, neurofilaments spend at most 20% of the time moving and rest of the time paused. These results establish that the slow axonal transport machinery conveys neurofilaments.

Adrenergic Fibers↗
PubMed2001

Amino acid transporters involved in luminal transport of mercuric conjugates of cysteine in rabbit proximal tubule.

The primary aim of the present study was to test the hypothesis that amino acid transport systems are involved in absorptive transport of dicysteinylmercury (cysteine-Hg-cysteine). Luminal disappearance flux [JD, fmol x min(-1) (mm tubular length)(-1)] of inorganic mercury (Hg2+), in the form of dicysteinylmercury, was measured in isolated perfused S2 segments with various amino acids or amino acid analogs in the luminal compartment under one of two conditions, in the presence or absence of Na+. The control perfusion fluid contained 20 microM dicysteinylmercury. Replacing Na+ in both the bathing and perfusing solutions with N-methyl-D-glucamine reduced the JD of Hg2+ by about 40%. Nine amino acids and two amino acid analogs were coperfused individually (at millimolar concentrations) with dicysteinylmercury. The amino acids and amino acid analogs that had the greatest effect on the JD of Hg2+ were L-cystine, L-serine, L-histidine, L-tryptophan, and 2-(-)-endoamino-bicycloheptane-2-carboxylic acid. The greatest reduction (76%) in the total JD of Hg2+ occurred when L-cystine was coperfused with dicysteinylmercury in the presence of Na+. Overall, the current findings indicate that Hg2+ is transported from the lumen into proximal tubular epithelial cells via amino acid transporters that recognize dicysteinylmercury. In addition, the data indicate that multiple amino acid transporters are involved in the luminal uptake of dicysteinylmercury, including the Na+-dependent low-affinity L-cystine, B(0), and ASC systems and the Na+-independent L-system. Furthermore, the transport data obtained when L-cystine was added to the luminal fluid indicate strongly that dicysteinylmercury is likely transported as a molecular homolog of L-cystine.

Amino Acid Transport Systems↗
PubMed2001

Trafficking-dependent and -independent pathways of neurotransmitter transporter regulation differentially involving p38 mitogen-activated protein kinase revealed in studies of insulin modulation of norepinephrine transport in SK-N-SH cells.

Presynaptic, cocaine- and antidepressant-sensitive norepinephrine (NE) transporters (NETs) dictate levels of extracellular NE after vesicular release. Recent studies suggest that G protein-coupled receptors linked to protein kinase C (PKC) down-regulate cell surface NET protein levels and diminish NE uptake capacity. We identified distinct phosphatidylinositol 3-OH kinase (PI3K)-linked pathways supporting basal and insulin-triggered NE transport in the human noradrenergic neuroblastoma, SK-N-SH. Acute (0-60 min) insulin treatments produced a time- and concentration-dependent stimulation of NE transport, resolved in kinetic studies as an enhancement of NE transport capacity (Vmax) without an alteration in NE Km. Basal and insulin-modulated NET activities were reduced by the tyrosine kinase inhibitor genistein and the PI3K inhibitors wortmannin and LY-294002, but not by the PKC inhibitor staurosporine. PI3K activation was found to support phosphorylation of p38 mitogen-activated protein kinase (p38 MAPK). However, basal and insulin-stimulated NET activities were differentiated by their reliance on p38 MAPK activation. Thus, the p38 MAPK inhibitor SB203580 and SB202190 abolished insulin activation of NE transport yet failed to impact basal NET activity. Moreover, p38 MAPK activation and insulin activation of NETs were found to be sensitive to external Ca2+ depletion, blockade of voltage-sensitive Ca2+ channels, and inhibition of protein phosphatase 2A. Effects of tyrosine kinase and PI3K inhibitors on basal NET uptake appear to arise from a loss of cell surface NET protein, whereas the p38 MAPK-dependent enhancement of NE transport occurs without a detectable enhancement of surface NET. Our findings establish two distinct pathways for regulation of NE uptake involving PI3K, one linked to transporter trafficking and a second linked to Ca2+-dependent, p38 MAPK phosphorylation that promotes activation of cell surface NETs.

Blotting, Western↗
PubMed1990

Human facilitative glucose transporters. Isolation, functional characterization, and gene localization of cDNAs encoding an isoform (GLUT5) expressed in small intestine, kidney, muscle, and adipose tissue and an unusual glucose transporter pseudogene-like sequence (GLUT6).

Two novel facilitative glucose transporter-like cDNAs have been isolated from human small intestine and fetal skeletal muscle cDNA libraries by low stringency cross-hybridization with a fragment of the human erythrocyte/GLUT1 facilitative glucose transporter cDNA. One encodes a 501-amino acid facilitative glucose transporter, designated as the small intestine/GLUT5 isoform, having 41.7, 40.0, 38.7, and 41.6% identity with the previously described human erythrocyte/GLUT1, liver/GLUT2, brain/GLUT3, and muscle-fat/GLUT4 isoforms, respectively. GLUT5 mRNA is expressed at highest levels in small intestine and at much lower levels in kidney, skeletal muscle, and adipose tissue. Expression of in vitro synthesized human GLUT5 mRNA in Xenopus laevis oocytes indicates that the GLUT5 protein is a cytochalasin B-sensitive glucose carrier. The gene encoding the GLUT5 protein is located on the short arm of human chromosome 1. The second facilitative transporter-like cDNA sequence, designated GLUT6, is part of an 11-kilobase transcript that is expressed in all tissues examined. The sequence of a partial-length GLUT6 cDNA having an insert of 3.4 kilobase pairs revealed a region of 1.5 kilobase pairs that has 79.6% identity with the human brain/GLUT3 facilitative glucose transporter cDNA. However, because of the presence of multiple stop codons and frame shifts, this sequence cannot encode a functional glucose transporter protein. The region of facilitative glucose transporter nucleotide sequence homology in the GLUT6 transcript may have arisen by insertion of a reverse-transcribed GLUT3 transcript into the untranslated region of another gene. The GLUT6 gene is located on the long arm of human chromosome 5.

Adipose Tissue↗