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At least 1,009 records · Page 56Linked to original sources
PubMed1993

Glucose transport activity and photolabelling with 3-[125I]iodo-4-azidophenethylamido-7-O-succinyldeacetyl (IAPS)-forskolin of two mutants at tryptophan-388 and -412 of the glucose transporter GLUT1: dissociation of the binding domains of forskolin and glucose.

The tryptophan residues 388 and 412 in the glucose transporter GLUT1 were altered to leucine (L) by site-directed mutagenesis and were transiently expressed in COS-7 cells. As assessed by immunoblotting, comparable numbers of glucose transporters were present in plasma membranes from cells transfected with wild-type GLUT1, GLUT1-L388 or GLUT1-L412. Transfection of the wild-type GLUT1 gave rise to a 3-fold increase in the reconstituted glucose transport activity recovered from plasma membranes. In contrast, transfection of GLUT1-L412 failed to increase the reconstituted transport activity, whereas transfection of GLUT1-L388 produced only a 70% increase. Photolabelling of GLUT1-L412 with 3-[125I]iodo-4-azidophenethylamido-7-O-succinyldeacetyl (125IAPS)-forskolin was not different from that of the wild-type GLUT1, whereas the GLUT1-L388 incorporated 70% less photolabel than did the wild-type GLUT1. These data suggest a dissociation of the binding sites of forskolin and glucose in GLUT1. Whereas both tryptophan-388 and tryptophan-412 appear indispensable for the function of the transporter, only tryptophan-388 is involved in the binding of the inhibitory ligand forskolin.

Affinity Labels↗
PubMed1996

Overexpression of catalytic subunit p110alpha of phosphatidylinositol 3-kinase increases glucose transport activity with translocation of glucose transporters in 3T3-L1 adipocytes.

To elucidate the mechanisms of phosphatidylinositol (PI) 3-kinase involvement in insulin-stimulated glucose transport activity, the epitope-tagged p110alpha subunit of PI 3-kinase was overexpressed in 3T3-L1 adipocytes using an adenovirus-mediated gene transduction system. Overexpression of p110alpha was confirmed by immunoblot using anti-tagged epitope antibody. p110alpha overexpression induced a 2.5-fold increase in PI 3-kinase activity associated with its regulatory subunits in the basal state, an increase exceeding that of the maximally insulin-stimulated control cells, while PI 3-kinase activity associated with phosphotyrosyl protein was only modestly elevated. Overexpression of p110alpha induced an approximately 14-fold increase in the basal glucose transport rate, which was also greater than that observed in the stimulated control. No apparent difference was observed in the cellular expression level of either GLUT1 or GLUT4 proteins between control and p110alpha-overexpressing 3T3-L1 adipocytes. Subcellular fractionation revealed translocation of glucose transporters from intracellular to plasma membranes in basal p110alpha-overexpressing cells. The translocation of GLUT4 protein to the plasma membrane was further confirmed using a membrane sheet assay. These findings indicate that an increment in PI 3-kinase activity induced by overexpression of p110alpha of PI 3-kinase stimulates glucose transport activity with translocation of glucose transporters, i.e., mimics the effect of insulin.

3T3 Cells↗
PubMed1998

Molecular characterization and tissue distribution of a new organic anion transporter subtype (oatp3) that transports thyroid hormones and taurocholate and comparison with oatp2.

Two complementary DNAs for the organic anion transporter subtypes oatp2 and oatp3, which transport thyroid hormones as well as taurocholate, were isolated from a rat retina cDNA library. The sequence of oatp2 is identical to that recently reported (Noé, B., Hagenbuch, B., Stieger, B., and Meier, P. J. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 10346-10350), whereas the sequence of oatp3 is novel. oatp3 consists of 670 amino acid residues and exhibits a structural architecture common to the organic anion transporter family, possessing the 12 putative membrane-spanning segments. Oocytes injected with oatp2 and oatp3 cRNAs showed taurocholate uptake in a saturable manner. The oatp2 and oatp3 cRNA-injected oocytes also showed significant uptake of both thyroxine and triiodothyronine. Northern blot and in situ analyses showed that the oatp2 mRNA was widely expressed in neuronal cells of the central nervous system, especially in the hippocampus, cerebellum, and choroid plexus as well as in the retina and liver. The oatp3 mRNA was highly expressed in the kidney and moderately abundant in the retina. This suggests that oatp2 and oatp3 are multifunctional transporters involved in the transport of thyroid hormones in the brain, retina, liver, and kidney.

Amino Acid Sequence↗
PubMed2000

Mechanism of electrogenic cation transport by the cloned organic cation transporter 2 from rat.

The organic cation transporter 2 (OCT2) is expressed in plasma membranes of kidney and brain. Its transport mechanism and substrates are debated. We studied substrate-induced changes of electrical current with the patch clamp technique after expression of rat OCT2 in oocytes. Activation of current, corresponding to efflux, was observed for small organic cations, e.g. choline. In contrast, the bigger cations quinine and tetrabutylammonium elicited no change in current. However, transport of choline could be inhibited by applying quinine or tetrabutylammonium to the cytoplasmic side. Inhibition of organic cation efflux by quinine was competitive with substrates. Quinine at the inside also inhibited substrate influx from the outside. Current-voltage analysis showed that both maximal turnover and apparent affinity to substrates are voltage-dependent. Substrate-induced currents with organic cations on both membrane sides reversed as predicted from the Nernst potential. Our results clearly identify the electrochemical potential as driving force for transport at neutral pH and exclude an electroneutral H(+)/organic cation(+) exchange. We suggest the existence of an electroneutral organic cation(+) exchange and propose a model for a carrier-type transport mechanism.

Animals↗
PubMed2000

The role of N-glycosylation in transport to the plasma membrane and sorting of the neuronal glycine transporter GLYT2.

Glycine transporter GLYT2 is an axonal glycoprotein involved in the removal of glycine from the synaptic cleft. To elucidate the role of the carbohydrate moiety on GLYT2 function, we analyzed the effect of the disruption of the putative N-glycosylation sites on the transport activity, intracellular traffic in COS cells, and asymmetrical distribution of this protein in polarized Madin-Darby canine kidney (MDCK) cells. Transport activity was reduced by 35-40% after enzymatic deglycosylation of the transporter reconstituted into liposomes. Site-directed mutagenesis of the four glycosylation sites (Asn-345, Asn-355, Asn-360, and Asn-366), located in the large extracellular loop of GLYT2, produced an inactive protein that was retained in intracellular compartments when transiently transfected in COS cells or in nonpolarized MDCK cells. When expressed in polarized MDCK cells, wild type GLYT2 localizes in the apical surface as assessed by transport and biotinylation assays. However, a partially unglycosylated mutant (triple mutant) was distributed in a nonpolarized manner in MDCK cells. The apical localization of GLYT2 occurred by a glycolipid rafts independent pathway.

Amino Acid Transport Systems, Neutral↗
PubMed2001

Ventricular choline transport: a role for organic cation transporter 2 expressed in choroid plexus.

To determine whether organic cation transporter (OCT) family members might mediate choline transport in choroid plexus (CP), the handling of choline by cloned transporters and by intact CP isolated from the adult rat was investigated. Expression of OCT1 and OCT2 in Xenopus oocytes increased hemicholinium-3-sensitive choline uptake. In contrast, OCT3 did not mediate choline transport. Estimated K(m) values for choline in rOCT1-, rOCT2-, and hOCT2-expressing oocytes were 346 +/- 50, 441 +/- 67, and 102 +/- 80 microm, respectively. Membrane potential was the major driving force for choline uptake in rat and human OCT2-expressing oocytes and in intact CP in vitro. Lowering of medium pH (6 versus 7.4) was equally effective at inhibiting choline uptake in CP, suggesting that there might be a non-OCT component of choline uptake that is responsive to an H(+) gradient. However, choline efflux from CP was not stimulated by a trans-applied H(+) gradient. Choline uptake by CP was Na(+)-independent with an estimated K(m) of 183 microm. Reverse transcriptase-polymerase chain reaction detected OCT2 and OCT3, but not OCT1, mRNA expression in CP. Transfection of intact CP with a rOCT2/green fluorescent protein fusion construct resulted in strong apical membrane fluorescence with no detectable signal in the basal and lateral plasma membranes. These data indicate that OCT2 mediates choline transport across the ventricular membrane of CP.

Animals↗
PubMed2003

The role of FhuD2 in iron(III)-hydroxamate transport in Staphylococcus aureus. Demonstration that FhuD2 binds iron(III)-hydroxamates but with minimal conformational change and implication of mutations on transport.

The fhuD2 gene encodes a lipoprotein that has previously been shown to be important for the utilization of iron(III)-hydroxamates by Staphylococcus aureus. We have studied the function of the FhuD2 protein in greater detail, and demonstrate here that the protein binds several iron(III)-hydroxamates. Mutagenesis of FhuD2 identified several residues that were important for the ability of the protein to function in iron(III)-hydroxamate transport. Several residues, notably Tyr-191, Trp-197, and Glu-202, were found to be critical for ligand binding. Moreover, mutation of two highly conserved glutamate residues, Glu-97 and Glu-231, had no affect on ligand binding, but did impair iron(III)-hydroxamate transport. Interestingly, the transport defect was not equivalent for all iron(III)-hydroxamates. We modeled FhuD2 against the high resolution structures of Escherichia coli FhuD and BtuF, two structurally related proteins, and showed that the three proteins share a similar overall structure. FhuD2 Glu-97 and Glu-231 were positioned on the surface of the N and C domains, respectively. Characterization of E97A, E231A, or E97A/E231A mutants suggests that these residues, along with the ligand itself, play a cumulative role in recognition by the ABC transporter FhuBGC2. In addition, small angle x-ray scattering was used to demonstrate that, in solution, FhuD2 does not undergo a detectable change in conformation upon binding iron(III)-hydroxamates. Therefore, the mechanism of binding and transport of ligands for binding proteins within this family is significantly different from that of other well studied binding protein families, such as that represented by maltose-binding protein.

Amino Acid Sequence↗
PubMed2004

Activation of syntaxin 1C, an alternative splice variant of HPC-1/syntaxin 1A, by phorbol 12-myristate 13-acetate (PMA) suppresses glucose transport into astroglioma cells via the glucose transporter-1 (GLUT-1).

Syntaxin 1C is an alternative splice variant lacking the transmembrane domain of HPC-1/syntaxin 1A. We found previously that syntaxin 1C is expressed as a soluble protein in human astroglioma (T98G) cells, and syntaxin 1C expression is enhanced by stimulation with phorbol 12-myristate 13-acetate (PMA). However, the physiological function of syntaxin 1C is not known. In this study, we examined the relationship between syntaxin 1C and glucose transport. First, we discovered that glucose transporter-1 (GLUT-1) was the primary isoform in T98G cells. Second, we demonstrated that glucose uptake in T98G cells was suppressed following an increase in endogenous syntaxin 1C after stimulation with PMA, which did not alter the expression levels of other plasma membrane syntaxins. We further examined glucose uptake and intracellular localization of GLUT-1 in cells that overexpressed exogenous syntaxin 1C; glucose uptake via GLUT-1 was inhibited without affecting sodium-dependent glucose transport. The value of Vmax for the dose-dependent uptake of glucose was reduced in syntaxin 1C-expressing cells, whereas there was no change in Km. Immunofluorescence studies revealed a reduction in the amount of GLUT-1 in the plasma membrane in cells that expressed syntaxin 1C. Based on these results, we postulate that syntaxin 1C regulates glucose transport in astroglioma cells by changing the intracellular trafficking of GLUT-1. This is the first report to indicate that a syntaxin isoform that lacks a transmembrane domain can regulate the intracellular transport of a plasma membrane protein.

Alternative Splicing↗
PubMed2004

Partitioning of the serotonin transporter into lipid microdomains modulates transport of serotonin.

The serotonin transporter (SERT) is an integral membrane protein responsible for the clearance of serotonin from the synaptic cleft following the release of the neurotransmitter. SERT plays a prominent role in the regulation of serotoninergic neurotransmission and is a molecular target for multiple antidepressants as well as substances of abuse. Here we show that SERT associates with lipid rafts in both heterologous expression systems and rat brain and that the inclusion of the transporter into lipid microdomains is critical for serotonin uptake activity. SERT is present in a subpopulation of lipid rafts, which is soluble in Triton X-100 but insoluble in other non-ionic detergents such as Brij 58. Disaggregation of lipid rafts upon depletion of cellular cholesterol results in a decrease of serotonin transport capacity (V(max)), due to the reduction of turnover number of serotonin transport. Our data suggest that the association of SERT with lipid rafts may represent a mechanism for regulating the transporter activity and, consequently, serotoninergic signaling in the central nervous system, through the modulation of the cholesterol content in the cell membrane. Furthermore, SERT-containing rafts are detected in both intracellular and cell surface fractions, suggesting that raft association may be important for trafficking and targeting of SERT.

Animals↗
PubMed2006

Transmembrane segment 12 of the Glut1 glucose transporter is an outer helix and is not directly involved in the transport mechanism.

A model has been proposed for the exofacial configuration of the Glut1 glucose transporter in which eight transmembrane domains form an inner helical bundle stabilized by four outer helices. The role of transmembrane segment 12, predicted to be an outer helix in this hypothetical model, was examined by cysteine-scanning mutagenesis and the substituted cysteine accessibility method using the membrane-impermeant, sulfhydryl-specific reagent, p-chloromercuribenzenesulfonate (pCMBS). A previously characterized functional cysteine-less Glut1 molecule was used to produce 21 Glut1 point mutants by changing each residue along helix 12 to a cysteine residue. These mutants were then expressed in Xenopus oocytes, and their protein levels, functional activities, and sensitivities to pCMBS were determined. Strikingly, in contrast to all nine other predicted Glut1 transmembrane helices that have been previously examined by this method, none of the 21 helix 12 single-cysteine mutants exhibited significant inhibition of specific transport activity. Also unlike most other Glut1 transmembrane domains in which solvent-accessible residues lie along a single face of the helix, mutations in five consecutive residues predicted to lie close to the exofacial face of the membrane resulted in sensitivity to pCMBS-induced transport inhibition. These results suggest that helix 12 plays a passive stabilizing role in the structure of Glut1 and is not directly involved in the transport mechanism. Additionally, the pCMBS data indicate that the predicted exoplasmic end of helix 12 is completely exposed to the external solvent when the transporter is in its exofacial configuration.

4-Chloromercuribenzenesulfonate↗
PubMed1997

Thrombin stimulates glucose transport in human platelets via the translocation of the glucose transporter GLUT-3 from alpha-granules to the cell surface.

Increased energy metabolism in the circulating blood platelet plays an essential role in platelet plug formation and clot retraction. This increased energy consumption is mainly due to enhanced anaerobic consumption of glucose via the glycolytic pathway. The aim of the present study was to determine the role of glucose transport as a potential rate-limiting step for human platelet glucose metabolism. We measured in isolated platelet preparations the effect of thrombin and ADP activation, on glucose transport (2-deoxyglucose uptake), and the cellular distribution of the platelet glucose transporter (GLUT), GLUT-3. Thrombin (0.5 U/ml) caused a pronounced shape change and secretion of most alpha-granules within 10 min. During that time glucose transport increased approximately threefold, concomitant with a similar increase in expression of GLUT-3 on the plasma membrane as observed by immunocytochemistry. A major shift in GLUT-3 labeling was observed from the alpha-granule membranes in resting platelets to the plasma membrane after thrombin treatment. ADP induced shape change but no significant alpha-granule secretion. Accordingly, ADP-treated platelets showed no increased glucose transport and no increased GLUT-3 labeling on the plasma membrane. These studies suggest that, in human blood platelets, increased energy metabolism may be precisely coupled to the platelet activation response by means of the translocation of GLUT-3 by regulated secretion of alpha-granules. Observations in megakaryocytes and platelets freshly fixed from blood confirmed the predominant GLUT-3 localization in alpha-granules in the isolated cells, except that even less GLUT-3 is present at the plasma membrane in the circulating cells (approximately 15%), indicating that glucose uptake may be upregulated five to six times during in vivo activation of platelets.

Adenosine Diphosphate↗
PubMed2001

Increased expression of neuronal glucose transporter 3 but not glial glucose transporter 1 following severe diffuse traumatic brain injury in rats.

Traumatic brain injury results in an increased brain energy demand that is associated with profound changes in brain glycolysis and energy metabolism. Increased glycolysis must be met by increasing glucose supply that, in brain, is primarily mediated by two members of the facilitative glucose transporter family, Glut1 and Glut3. Glut1 is expressed in endothelial cells of the blood-brain barrier (BBB) and also in glia, while Glut3 is the primary glucose transporter expressed in neurons. However, few studies have investigated the changes in glucose transporter expression following traumatic brain injury, and in particular, the neuronal and glial glucose transporter responses to injury. This study has therefore focussed on investigating the expression of the glial specific 45-kDa isoform of Glut1 and neuronal specific Glut3 following severe diffuse traumatic brain injury in rats. Following impact-acceleration injury, Glut3 expression was found to increase by at least 300% as early as 4 h after induction of injury and remained elevated for at least 48 h postinjury. The increase in Glut3 expression was clearly evident in both the cerebral cortex and cerebellum. In contrast, expression of the glial specific 45-kDa isoform of Glut1 did not significantly change in either the cerebral cortex or cerebellum following traumatic injury. We conclude that increased glucose uptake after traumatic brain injury is primarily accounted for by increased neuronal Glut 3 glucose transporter expression and that this increased expression after trauma is part of a neuronal stress response that may be involved in increasing neuronal glycolysis and associated energy metabolism to fuel repair processes.

Animals↗
PubMed2004

Mutational scanning of the human serotonin transporter reveals fast translocating serotonin transporter mutants.

The serotonin transporter (SERT) belongs to a family of sodium-chloride-dependent transporters responsible for uptake of amino acids and biogenic amines from the extracellular space. SERT represents a major pharmacological target in the treatment of several clinical conditions, including depression and anxiety. In the present study we have undertaken a mutational scanning of human SERT in order to identify residues that are responsible for individual differences among related monoamine transporters. One mutant, G100A, was inactive in transport. However, ligand binding affinity was similar to wild-type, suggesting that G100A amongst different possible SERT conformations is restrained to a binding conformation. We suggest that the main role of glycine-100 is to confer structural flexibility during substrate translocation. For the two single mutants, T178A and F263C, uptake rates and K(m) values were both several-fold higher than wild-type while binding affinities and inhibitory potencies decreased considerably for several drugs. Ion dependency increased and only at hyperosmotic concentrations were K(m) values partly restored. For the double mutant, T178A/F263C, shifts in uptake kinetics and ligand affinities, as well as ion dependencies, were drastic. Effects were synergistic compared to the corresponding single mutants. In conclusion, we suggest that mutating threonine-178 to an alanine and phenylalanine-263 to a cysteine mainly alter the overall uptake kinetics of SERT by affecting the conformational equilibrium of different transporter conformations.

Amino Acid Sequence↗
PubMed2005

The role of N-glycosylation in the stability, trafficking and GABA-uptake of GABA-transporter 1. Terminal N-glycans facilitate efficient GABA-uptake activity of the GABA transporter.

Neurotransmitter transporters play a major role in achieving low concentrations of their respective transmitter in the synaptic cleft. The GABA transporter GAT1 belongs to the family of Na(+)- and Cl(-)-coupled transport proteins which possess 12 putative transmembrane domains and three N-glycosylation sites in the extracellular loop between transmembrane domain 3 and 4. To study the significance of N-glycosylation, green fluorescence protein (GFP)-tagged wild type GAT1 (NNN) and N-glycosylation defective mutants (DDQ, DGN, DDN and DDG) were expressed in CHO cells. Compared with the wild type, all N-glycosylation mutants showed strongly reduced protein stability and trafficking to the plasma membrane, which however were not affected by 1-deoxymannojirimycin (dMM). This indicates that N-glycosylation, but not terminal trimming of the N-glycans is involved in the attainment of a correctly folded and stable conformation of GAT1. All N-glycosylation mutants were expressed on the plasma membrane, but they displayed markedly reduced GABA-uptake activity. Also, inhibition of oligosaccharide processing by dMM led to reduction of this activity. Further experiments showed that both N-glycosylation mutations and dMM reduced the V(max) value, while not increasing the K(m) value for GABA uptake. Electrical measurements revealed that the reduced transport activity can be partially attributed to a reduced apparent affinity for extracellular Na+ and slowed kinetics of the transport cycle. This indicates that N-glycans, in particular their terminal trimming, are important for the GABA-uptake activity of GAT1. They play a regulatory role in the GABA translocation by affecting the affinity and the reaction steps associated with the sodium ion binding.

1-Deoxynojirimycin↗
PubMed2006

Monocarboxylate transporter 1 (MCT1) mediates transport of short-chain fatty acids in bovine caecum.

The present study was undertaken to investigate the functional role of monocarboxylate transporter 1 (MCT1) in the ruminant large intestine. Messenger RNA encoding for MCT1 was verified by reverse transcriptase-polymerase chain reaction in caecum, proximal colon and distal colon of adult cattle. Both immunohistochemistry and confocal laser microscopy verified that the MCT1 protein was abundant in the surface epithelium of the large intestine, and the amount decreased from the opening of the crypt to its base. In the immunopositive cells, MCT1 was primarily localized in the basolateral membranes of epithelium lining the large intestine. Western blotting indicated that the levels of MCT1 protein were highest in the caecum, followed by proximal colon and then distal colon. In vitro studies were conducted to elucidate the possible involvement of MCT1 in the transport of short-chain fatty acids (SCFA) across the isolated mucosal sheets of cattle caecum using the Ussing chamber technique. Acetate absorption was found to be pH dependent, and the rate of acetate absorption increased as pH decreased. The serosal application of the MCT1 inhibitor 'p-chloromercuribenzoic acid (pCMB)' significantly reduced the transport of acetate across the caecal epithelium of cows. In addition, the transport of acetate was significantly reduced in the presence of its analogue, propionate, indicating that acetate and propionate compete for binding to the same transporter. The results show that MCT1 is a major route for SCFA efflux across the basolateral membrane of bovine large intestine and that it could play a role in the regulation of intracellular pH.

Acetates↗
PubMed2006

Testosterone increases lactate transport, monocarboxylate transporter (MCT) 1 and MCT4 in rat skeletal muscle.

We have examined the effects of administration of testosterone for 7 days on monocarboxylate transporter (MCT) 1 and MCT4 mRNAs and proteins in seven metabolically heterogeneous rat hindlimb muscles and in the heart. In addition, we also examined the effects of testosterone treatment on plasmalemmal MCT1 and MCT4, and lactate transport into giant sarcolemmal vesicles prepared from red and white hindlimb muscles and the heart. Testosterone did not alter MCT1 or MCT4 mRNA, except in the plantaris muscle. Testosterone increased MCT1 (20%-77%, P < 0.05) and MCT4 protein (29%-110%, P< 0.05) in five out of seven muscles examined. In contrast, in the heart MCT1 protein was not increased (P> 0.05), and MCT 4 mRNA and protein were not detected. There was no correlation between the testosterone-induced increments in MCT1 and MCT4 proteins. Muscle fibre composition was not associated with testosterone-induced increments in MCT1 protein. In contrast, there was a strong positive relationship between the testosterone-induced increments in MCT4 protein and the fast-twitch fibre composition of rat muscles. Lactate transport into giant sarcolemmal vesicles was increased in red (23%, P< 0.05) and white muscles (21%, P< 0.05), and in the heart (58%, P< 0.05) of testosterone-treated animals (P< 0.05). However, plasmalemmal MCT1 protein (red, +40%, P< 0.05; white, +39%, P< 0.05) and plasmalemmal MCT4 protein (red, +25%, P< 0.05; white, +48%, P< 0.05) were increased only in skeletal muscle. In the heart, plasmalemmal MCT1 protein was reduced (-20%, P< 0.05). In conclusion, these studies have shown that testosterone induces an increase in both MCT1 and MCT4 proteins and their plasmalemmal content in skeletal muscle. However, the testosterone-induced effect was tissue-specific, as MCT1 protein expression was not altered in the heart. In the heart, the testosterone-induced increase in lactate transport cannot be explained by changes in plasmalemmal MCT1 content, but in skeletal muscle the increase in the rate of lactate transport was associated with increases in plasmalemmal MCT1 and MCT4.

Animals↗
PubMed1992

Glucose transporters and maximal transport are increased in endurance-trained rat soleus.

Voluntary wheel running induces an increase in the concentration of the regulatable glucose transporter (GLUT4) in rat plantaris muscle but not in soleus muscle (K. J. Rodnick, J. O. Holloszy, C. E. Mondon, and D. E. James. Diabetes 39: 1425-1429, 1990). Wheel running also causes hypertrophy of the soleus in rats. This study was undertaken to ascertain whether endurance training that induces enzymatic adaptations but no hypertrophy results in an increase in the concentration of GLUT4 protein in rat soleus (slow-twitch red) muscle and, if it does, to determine whether there is a concomitant increase in maximal glucose transport activity. Female rats were trained by treadmill running at 25 m/min up a 15% grade, 90 min/day, 6 days/wk for 3 wk. This training program induced increases of 52% in citrate synthase activity, 66% in hexokinase activity, and 47% in immunoreactive GLUT4 protein concentration in soleus muscles without causing hypertrophy. Glucose transport activity stimulated maximally with insulin plus contractile activity was increased to roughly the same extent (44%) as GLUT4 protein content in soleus muscle by the treadmill exercise training. In a second set of experiments, we examined whether a swim-training program increases glucose transport activity in the soleus in the presence of a maximally effective concentration of insulin. The swimming program induced a 44% increase in immunoreactive GLUT4 protein concentration. Glucose transport activity maximally stimulated with insulin was 62% greater in soleus muscle of the swimmers than in untrained controls. Training did not alter the basal rate of 2-deoxyglucose uptake.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed2004

Novel slc22 transporter homologs in fly, worm, and human clarify the phylogeny of organic anion and cation transporters.

Slc22 family organic anion and cation transporters (OATs, OCTs, and OCTNs) are transmembrane proteins expressed predominantly in kidney and liver. These proteins mediate the uptake or excretion of numerous physiologically (and pharmacologically) important compounds, and accordingly have been the focus of intensive study. Here we investigate the molecular phylogeny of the slc22 transporters, identifying homologs in Drosophila and C. elegans, several of which are developmentally regulated, as well as reporting the cloning of a novel human family member, UST6, expressed exclusively in liver in both embryo and adult. The latter helps define a subfamily within the OATs, which appears to have human- and rodent-specific members, raising potential issues with respect to the use of rodents as models for the transport of organic anions (which include many pharmaceuticals) in humans. Although this phylogenetic inference could not be made on the basis of sequence alignment, analysis of intron phasing suggests that the OAT, OCT, and OCTN lineages of the slc22 family formed after the divergence of vertebrates and invertebrates. Subsequently, these lineages expanded through independent tandem duplications to produce multiple gene pairs. After analyzing over 200 other transporter genes, we find such pairing to be relatively specific to vertebrate organic anion and cation transporters, suggesting selection for gene pairing operating within this family in particular. This might reflect a requirement for redundancy or broader substrate specificity in vertebrates (compared to invertebrates), due to their greater physiological complexity and thus potentially broader exposure to organic ions.

Amino Acid Sequence↗