Konrad Keller

D-Glucose Transport in Cultured Cells of Neural Origin: The Membrane as Possible Control Point of Glucose Utilization

Abstract: The function of plasma membrane as control point of glucose metabolism has been studied in confluent monolayer of C1300 neuroblastoma (N2A) and glioma (C6) cells. In neuroblastoma, steady state intracellular glucose concentration reached the extracellular levels, while intracellular contents in C6 glioma cells remained very low. In C6 glial cells the amount of glycogen as source of energy was much higher than that found in C1300 neuroblastoma cells. Influx rates of D‐glucose in C6 glioma cells were only half those found in neuroblastoma cells. During the influx period (0‐40 s) the transport of glucose in these cells did not exceed the phosphorylation rate, whereas a steady, time‐dependent increase in glucose content was observed in neuroblastoma cells. While glucose uptake in neuroblastoma cells seems to be regulated at the level of phosphorylating enzymes, the control point in C6 glioma is believed to be membrane transport.

Estimation of water content and water mobility in the nucleus and cytoplasm of Xenopus laevis oocytes by NMR microscopy

NMR microscopy is a noninvasive approach for studying cell structure and properties. Spatially resolved measurements of the relaxation times T1 and T2 provided information on the water proton spin density and water mobility in different parts of Xenopus laevis oocytes. The spin-lattice relaxation time T1 was determined using a saturation-recovery sequence and the common spin-echo sequence with increasing repetition times, while the transverse relaxation time T2 was measured by means of the spin-echo sequence with varying echo times. From the relaxation times, the mole fractions of possible reorientational correlation times tau c for different types of intracellular water were calculated according to a simple two-phase model. The values for T1, T2, and proton spin density (i.e., water content) are: nucleus >> animal cytoplasm > vegetal cytoplasm. Based on the estimation of tau c, nearly 90% of the nuclear water and 74.4% of the water of the animal pole was considered as free mobile water, whereas 55.5% of the water of the vegetal pole appeared as bound water.

Subcellular distribution and activity of glucose transporter isoforms GLUT1 and GLUT4 transiently expressed in COS-7 cells.

In adipose and muscle cells, the glucose transporter isoform GLUT4 is mainly located in an intracellular, vesicular compartment from which it is translocated to the plasma membrane in response to insulin. In order to test the hypothesis that this preferential targeting of a glucose transporter to an intracellular storage site is conferred only by its primary sequence, we compared the subcellular distribution of the fat/muscle glucose transporter GLUT4 with that of the erythrocyte/brain-type glucose transporter GLUT1 after transient expression in COS-7 cells. Full-length cDNA was ligated into the expression vector pCMV that is driven by the cytomegalovirus promoter, and introduced into COS cells by the DEAE-dextran method. Cells were homogenized and fractionated by differential centrifugation to yield plasma membranes and a Golgi-enriched fraction of intracellular membranes (low-density microsomes). In these membrane fractions, the abundance of glucose transporters was assessed by immunoblotting with specific antibodies against GLUT1 and GLUT4, and their transport activity was assayed after solubilization and reconstitution into lecithin liposomes. Uptake rates of 2-deoxyglucose assayed in parallel samples were higher in cells expressing GLUT1 or GLUT4 as compared with control cells (transfection of pCMV without transporter cDNA). Reconstituted glucose transport activity in plasma membranes was about 5-fold higher after expression of GLUT1 and GLUT4 as compared with control cells. The relative amount of GLUT4 in the low-density microsomes as detected by reconstitution and immunoblotting exceeded that of the GLUT1, but was much lower than that observed in typical insulin-sensitive cells, e.g., rat fat cells or 3T3-L1 adipocytes. These data indicate that COS-7 cells transfected with glucose transporter cDNA express the active transport proteins and can be used for functional studies.

Association of glycolytic enzymes with the cytoplasmic side of the plasma membrane of glioma cells

A latex phagocytosis technique was used to prepare relatively pure plasma membranes with inside-out orientation. This method was adapted through a number of modifications in order to evaluate the association of glycolytic enzymes with the cytoplasmic side of the plasma membrane of C6 glial cells. As phosphorylation is strictly coupled with transport in these cells, glycolytic enzymes, especially hexokinase, could metabolize glucose in close vicinity to its transporter. Of the enzymes tested, hexokinase is present in considerable quantities on these membranes (nearly 40% of homogenate specific activity), followed by D-glyceraldehyde-3-phosphate dehydrogenase (10%), pyruvate kinase (8%), and 3-phosphoglycerate kinase (1%). Except for hexokinase, the enzyme pattern presented here is different from that published for other membrane preparations.

Glycolysis and glycogen metabolism after inhibition of hexose monophosphate pathway in C6-GLIAL cells.

Summary1.6-Aminonicotinamide (0.01 mg/ml) leads to a strong accumulation of 6-PG in C6 glial cells after 24 h.2.The accumulated 6-PG is dephosphorylated to gluconate which easily permeates the cell membrane. Extracellular gluconate is formed at a rate of 12% of the total glucose consumption.3.6-PG as competitive inhibitor of the PGI caused a reduction of the glycolytic flux of about 40%.4.The reduced glycolytic flux lowers the ATP concentration under anaerobic conditions to 75% of the controls.5.The glycogen content after 6-AN is increased by 50%, probably by the activation of the glycogen synthetase due to the higher Glc 6-P concentration.6.The fibroblast-like morphology of the C6 cell line has typically changed under 6-aminonicotinamide.

Functional consequences of the autosomal dominant G272A mutation in the human GLUT1 gene

The first autosomal dominant missense mutation (G272A) reported within the human GLUT1 gene and shared by three affected family members was investigated in respect to functional consequences. Substitution of glycine‐91 by site‐directed mutagenesis with either aspartate or alanine resulted in a significant decrease in transport activity of GLUT1 expressed in Xenopus oocytes. Expression of mutant transporters was confirmed by immunoblot, 2‐deoxy‐glucose uptake and confocal laser microscopy. The data agree with 3‐O‐methyl‐glucose uptake into patient erythrocytes and indicate that the loss of glycine rather than a hydrophilic side chain (Gly91Asp) defines the functional consequences of this mutation.

From triple cysteine mutants to the cysteine-less glucose transporter GLUT1: a functional analysis

Two triple cysteine mutants containing Cys‐less N‐ or C‐terminal halves and the Cys‐less GLUT1 were generated by site‐directed mutagenesis. Following expression in Xenopus oocytes, the intrinsic transport activities of the multiple cysteine mutants were slightly decreased when either the cysteine residues of the C‐terminal half or all six residues were changed; substitution of serine for cysteine residues located at the N‐terminal half was without consequence for the catalytic activity. The exofacial ligand ethylidene glucose inhibited 2‐deoxy‐d‐glucose uptake of wild‐type and Cys‐less GLUT1‐expressing Xenopus oocytes with comparable half‐saturation constants (11.5 and 13.2 mM). However, each of the multiple cysteine mutants exhibited an increase in affinity for the endofacial inhibitor cytochalasin B, with the greatest effect being observed for the Cys‐less construct (decrease in K i by the factor 5–6).

The differential role of Cys-421 and Cys-429 of the Glut1 glucose transporter in transport inhibition by p-chloromercuribenzenesulfonic acid (pCMBS) or cytochalasin B (CB)

Cys‐421 and Cys‐429 of Glut1 were replaced by site‐directed mutagenesis in order to investigate their involvement in basal glucose transport and transport inhibition. Neither of the two cysteine residues was essential for basal 2‐deoxy‐D‐glucose uptake in Xenopus oocytes expressing the respective mutant M421 and M429. If applied from the external side, the poorly permeable sulfhydryl‐reactive agent pCMBS inhibited 2‐deoxy‐D‐glucose uptake of Glut1 ‐ and M421‐expressing Xenopus oocytes but failed to affect uptake of the Cys‐429 mutant. This is in agreement with the proposed two‐dimensional model or Glut1 confirming that Cys‐429 is the only residue exposed to the surface of the plasma membrane. The replacement of Cys‐421 at the exofacial end of helix eleven caused a partial protection of 3‐O‐methylglucose transport inhibition by CB; this residue may thus be involved in stabilizing an adjacent local tertiary structure necessary for the full activity of this inhibitor.

Study of the membrane permeability of a paramagnetic metal complex on single cells by NMR microscopy

A new procedure has been developed for investigating the ability of paramagnetic metal complexes to penetrate the plasma membrane of eukaryotic cells without decomposition. Defolliculated Xenopus laevis oocytes formed the biological system to test N,N-ethylenebis-(1,5,5-trimethyltetramic-acid-3-acetiminato) copper (II). An increase of the signal intensities in spin-echo (SE) images of oocytes treated with the tested substance indicated that the complex was able to penetrate biological membranes due to the arrangement of hydrophobic and hydrophilic groups within the ligand. In contrast, the treatment with the commonly used contrast agent gadolinium-DTPA/dimeglumine did not enhance the signal intensity in NMR images of oocytes after time periods of exposure comparable to those used for the copper complex. After microinjection into Xenopus oocytes the copper complex was released into the extracellular medium without degradation, as shown by HPLC measurements.

Glucose metabolism in C-1300 neuroblastoma cells after inhibition of hexose monophosphate pathway.

Summary1.Application of 6-AN (0.01 mg/ml) leads to a strong accumulation of 6-PG in C-1300 neuroblastoma cells which, however, only amounts to one third of that found in C-6 glial cells.2.In C-1300 neuroblastoma cells dephosphorylation of the accumulated 6-PG causes a rise of the intracellular gluconate to eight times the value found for 6-PG. It is four times higher than the gluconate content observed in C-6 glical cells.3.Although 6-PG is a competitive inhibitor of PGI it causes no reduction of glycolytic flux and ATP content in stationary phase C-1300 neuroblastoma cells in contrast to the strong reduction of glycolytic flux and ATP content observed in C-glial cells.4.The intracellular Glc-6-P and Fru-6-P content of C-1300 neuroblastoma cells increases by four to five times after treatment with 6-AN. Both this increase and the decrease of Fru-1,6-P2 content point to an inhibition of the phosphofructokinase.5.In contrast to C-6 glial cells no morphological changes could be observed in C-1300 neuroblastoma cells up to 24 h after administration of 6-AN.