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Structural signatures and membrane helix 4 in GLUT1: inferences from human blood-brain glucose transport mutants.

ABSTRACT: Exon IV of SLC2A1, a multiple facilitator superfamily (MFS) transporter gene, is particularly susceptible to mutations that cause GLUT1 deficiency syndrome, a human encephalopathy that results from decreased glucose flux through the blood-brain barrier. Genotyping of 100 patients revealed that in a third of them who harbor missense mutations in the GLUT1 transporter, transmembrane domain 4 (TM4), encoded by SLC2A1 exon IV, contains mutant residues that have the periodicity of one face of a kinked alpha-helix. Arg-126, located at the amino terminus of TM4, is the locus for most of the mutations followed by other arginine and glycine residues located elsewhere in the transporter but conserved among MFS proteins. The Arg-126 mutants were constructed and assayed for protein expression, targeting, and transport capacity in Xenopus oocytes. The role of charge at position 126, as well as its accessibility, was investigated in R126H by determining its activity as a function of extracellular pH. The results indicate that intracellular charges at the MFS TM2-3 and TM8-9 signature loops and flanking TMs 3, 5, and 6 are critical for the structure of GLUT1 as are TM glycines and that TM4, located at the catalytic core of MFS proteins, forms a helix that surfaces into the extracellular solution where another proton facilitates transport.

SUBMITTER: Pascual JM

PROVIDER: S-EPMC2423257 | biostudies-literature | 2008 Jun

REPOSITORIES: biostudies-literature

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Structural signatures and membrane helix 4 in GLUT1: inferences from human blood-brain glucose transport mutants.

Pascual Juan M JM Wang Dong D Yang Ru R Shi Lei L Yang Hong H De Vivo Darryl C DC

The Journal of biological chemistry 20080403 24

Exon IV of SLC2A1, a multiple facilitator superfamily (MFS) transporter gene, is particularly susceptible to mutations that cause GLUT1 deficiency syndrome, a human encephalopathy that results from decreased glucose flux through the blood-brain barrier. Genotyping of 100 patients revealed that in a third of them who harbor missense mutations in the GLUT1 transporter, transmembrane domain 4 (TM4), encoded by SLC2A1 exon IV, contains mutant residues that have the periodicity of one face of a kinke ...[more]

PMID: 18387950

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Project description:BackgroundThyroid hormones (THs) act genomically to stimulate glucose transport by elevating glucose transporter (Slc2a) expression and glucose utilization by cells. However, nongenomic effects of THs are now emerging. Here, we assess how triiodothyronine (T(3)) acutely affects glucose transport and the content of GLUT4, GLUT1, and GLUT3 at the surface of muscle cells, and possible interactions between T(3) and insulin action.MethodsDifferentiated L6 myotubes transfected with myc-tagged Slc2a4 (L6-GLUT4myc) or Slc2a1 (L6-GLUT1myc) and wild-type L6 myotubes were studied in the following conditions: control, hypothyroid (Tx), Tx plus T(3), Tx plus insulin, and Tx plus insulin and T(3).ResultsGlucose uptake and GLUT4 content at the cell surface decreased in the Tx group relative to controls. T(3) treatment for 30 minutes increased glucose transport into L6-GLUT4myc cells without altering surface GLUT4 content, which increased only thereafter. The total amount of GLUT4 protein remained unchanged among the groups studied. The surface GLUT1 content of L6-GLUT1myc cells also remained unaltered after T(3) treatment; however, in these cells glucose transport was not stimulated by T(3). In wild-type L6 cells, although T(3) treatment increased the total amount of GLUT3, it did not change the surface GLUT3 content. Moreover, within 30 minutes, T(3) stimulation of glucose uptake was additive to that of insulin in L6-GLUT4myc cells. As expected, insulin elevated surface GLUT4 content and glucose uptake. However, interestingly, surface GLUT4 content remained unchanged or even dropped with T(3) plus insulin.ConclusionsThese data reveal that T(3) rapidly increases glucose uptake in L6-GLUT4myc cells, which, at least for 30 minutes, did not depend on an increment in GLUT4 at the cell surface yet potentiates insulin action. We propose that this rapid T(3) effect involves activation of GLUT4 transporters at the cell surface, but cannot discount the involvement of an unknown GLUT.

Project description:ObjectiveTo investigate if the role of Cav-1 in GDM-induced macrosomia is through regulating AMPK signaling pathway in placenta.MethodsWe used diagnostic criteria of gestational diabetes mellitus (GDM) and macrosomia to separate and compare placental protein and mRNA levels from GDM with macrosomia group (GDMM), GDM with normal birth weight group (GDMN) and normal glucose tolerance (NGT) with normal birth weight group (CON). Western blotting was performed to examine differentially expressed proteins of caveolin-1 (Cav-1) and Adenosine monophosphate-activated protein kinase (AMPK) signaling pathway related proteins, including phosphorylated-AMPK?(Thr172), AMPK?, phosphorylated-Acetyl-CoA carboxylase(Ser79) (p-ACC(Ser79)), ACC and glucose transporter 1 (GLUT1) in placenta between the three groups. The mRNA levels of Cav-1, AMPK?, ACC and GLUT1 in placenta were measured by real time-PCR.ResultsIn the GDMM placenta group, both protein and mRNA levels of Cav-1 were down-regulated, while GLUT1 was up-regulated; the phosphorylation and mRNA levels of ACC and AMPK? were decreased, but total ACC protein levels were increased compared to both the GDMN (p<0.05) and CON groups (p<0.05). In GDMM placenta group, there was a significant negative correlation observed between neonatal birth weight (NBW) and protein expression levels of Cav-1, p-ACC(Ser79) and p-AMPK?(Thr172) (p<0.05), while positive relationship with ACC and GLUT1 protein levels. Besides, in GDMM group placental mRNA levels, NBW had a positive correlation with GLUT1 (p<0.05), while negative with Cav-1, AMPK? and ACC expression (p<0.05). Cav-1 protein expression was positively associated with p-AMPK and p-ACC (p<0.05), and negatively associated with GLUT1 (p<0.05). Interestingly, p-AMPK protein expression was closely related to p-ACC (p<0.05), but not with GLUT1.ConclusionGDM-induced macrosomias have more severe inhibition of Cav-1 expression in placenta. Cav-1 is associated with placental glucose and fatty acid transport via the induction of AMPK signaling pathway and the reduction of GLUT1 signaling pathway to reverse GDM-induced macrosomia.

Project description:RATIONALE:Endothelial cells (ECs) are highly glycolytic and generate the majority of their energy via the breakdown of glucose to lactate. At the same time, a main role of ECs is to allow the transport of glucose to the surrounding tissues. GLUT1 (glucose transporter isoform 1/Slc2a1) is highly expressed in ECs of the central nervous system (CNS) and is often implicated in blood-brain barrier (BBB) dysfunction, but whether and how GLUT1 controls EC metabolism and function is poorly understood. OBJECTIVE:We evaluated the role of GLUT1 in endothelial metabolism and function during postnatal CNS development as well as at the adult BBB. METHODS AND RESULTS:Inhibition of GLUT1 decreases EC glucose uptake and glycolysis, leading to energy depletion and the activation of the cellular energy sensor AMPK (AMP-activated protein kinase), and decreases EC proliferation without affecting migration. Deletion of GLUT1 from the developing postnatal retinal endothelium reduces retinal EC proliferation and lowers vascular outgrowth, without affecting the number of tip cells. In contrast, in the brain, we observed a lower number of tip cells in addition to reduced brain EC proliferation, indicating that within the CNS, organotypic differences in EC metabolism exist. Interestingly, when ECs become quiescent, endothelial glycolysis is repressed, and GLUT1 expression increases in a Notch-dependent fashion. GLUT1 deletion from quiescent adult ECs leads to severe seizures, accompanied by neuronal loss and CNS inflammation. Strikingly, this does not coincide with BBB leakiness, altered expression of genes crucial for BBB barrier functioning nor reduced vascular function. Instead, we found a selective activation of inflammatory and extracellular matrix related gene sets. CONCLUSIONS:GLUT1 is the main glucose transporter in ECs and becomes uncoupled from glycolysis during quiescence in a Notch-dependent manner. It is crucial for developmental CNS angiogenesis and adult CNS homeostasis but does not affect BBB barrier function.

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Structural signatures and membrane helix 4 in GLUT1: inferences from human blood-brain glucose transport mutants.

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Structural signatures and membrane helix 4 in GLUT1: inferences from human blood-brain glucose transport mutants.

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