• BMB Reports
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• v.32 no.5
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• pp.429-435
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• 1999

Unlike the mammalian glucose transporter GLUT1, little is known about the nature of the endogenous sugar transporter(s) in insect cells. In order to establish the transport characteristics and other properties of the sugar transport proteins of Sf9 cells, a series of kinetic analyses was performed. A saturable transport system for hexose uptake has been revealed in the insect cells. The apparent affinity of this transport system(s) for 2-deoxy-D-glucose was relatively high, the $K_m$ for uptake being <0.5 mM. To further investigate the substrate and inhibitor recognition properties of the insect cell transporter, the ability of other sugars or drugs to inhibit 2-deoxy-D-glucose transport was examined by measuring inhibition constants ($K_j$). Transport was inhibited by D-mannose, D-glucose, and D-fructose. However, the apparent affinity of the C-4 epimer, D-galactose, for the Spodoptera transporter was relatively low, implying that the hydroxyl group at the C-4 position may play a role in the strong binding of glucose and mannose to the transporter. The results also showed that transport was stereoselective, being inhibited by D-glucose but not by L-glucose. It is therefore concluded that insect cells contain an endogenous glucose transport activity that in several aspects resembles the human erythrocyte glucose transporter. However, the mammalian and insect transporters were different in some of their kinetic properties, namely, their affinities for fructose and for cytochalasin B.

• The Korean Journal of Physiology
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• v.24 no.2
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• pp.331-344
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• 1990

What makes glucose transport function sensitive to insulin in one cell type such as adipocyte, and insensitive in another such as liver cells is unresolved question at this time. Recently it is known that insulin stimulates glucose transport in adipocytes largely by redistributing transporter from the storage pool that is included in a low density microsomal fraction to plasma membrane. Therefore, insulin sensitivity may depend upon the relative distribution of gluscose transporters between the plasma membrane and in an intracellular storage compartment. In hepatocytes, the subcellular distribution of glucose transporter is less well documented. It is thus possible that the apparent insensitivity of the hepatocyte system could be either due to lack of the constitutively maintained, intracellular storage pool of glucose transporter or lack of insulin-mediated transporter translocation mechanism in this cell. In this study, I examined if any intracellular glucose transporter pool exists in hepatocytes and this pool is affected by insulin. The results obtained summarized as followings: 1) Distribution of subcellular fractions of hepatocyte showed that there are $24.9{\pm}1.3%$ of plasma membrane, $36.9{\pm}1.7%$ of nucleus-mitochondria enriched fraction, $23.5{\pm}1.2%$ of lysosomal fraction, $9.6{\pm}1.0%$ of high density microsomal fraction and $4.9{\pm}0.5%$ of low density microsomal fraction. 2) In adipocyte, there were $29.9{\pm}2.6%$ of plasma membrane, $19.4{\pm}1.9%$ of nucleus-mitochondria enriched fraction, $26.7{\pm}1.8%$ of high density microsomal fraction and $23.9{\pm}2.1%$ of low density microsomal fraction. 3) Surface labelling of sodium borohydride revealed that plasma membrane contaminated to lysosomal fraction by $26.8{\pm}2.8%$, high density microsomal fraction by $8.3{\pm}1.3%$ and low density microsomal fraction by $1.7{\pm}0.4%$ respectively. 4) Cytochalasin B bound to all of subcellular fractions with a Kd of $1.0{\times}10^{-6}M$. 5) Photolabelling of cytochalasin B to subcellular fractions occurred on 45 K dalton protein band, a putative glucose transporter and D-glucose inhibited the photolabelling. 6) Insulin didn't affect on the distribution of subcellular fractions and translocation of intracellular glucose transporters of hepatocytes. 7) HEGT reconstituted into hepatocytes was largely associated with plasma membrane and very little was found in low density microsomal fraction which equals to the native glucose transporter distribution. Insulin didn't affect on the distribution of exogeneous glucose transporter in hepatocytes. From the above results it is concluded that insulin insensitivity of hepatocyte may due to lack of intracellular storage pool of glucose transporter and thus intracellular storage pool of glucose transporter is an essential feature of the insulin action.

• The Korean Journal of Physiology
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• v.27 no.2
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• pp.123-138
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• 1993

The mechanism of insulin action to increase glucose transport is attributed to glucose transporter translocation from intracellular storage pools to the plasma membrane in insulin-sensitive cells. The present study was designed to visualize the redistribution of the glucose transporter by means of an immunogold labelling method. Our data clearly show that glucose transporter molecules were visible by this method. According to the method this distribution of glucose transporters between cell surface and intracellular pool was different in adipocytes. The glucose transporter molecules were randomly distributed at the cell surface whereas the molecules at LDM were farmed as clusters. By insulin treatment the number of homogeneous random particles increased at the cell surface whereas the cluster forms decreased at the intracellular storage pools. It suggests that the active molecules needed to be evenly distributed far effective function and that the inactive molecules in storage pools gathered and termed clusters until being transferred to the plasma membrane.

• Biomedical Science Letters
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• v.9 no.4
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• pp.177-181
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• 2003

The baculovirus/Spodoptera frugiperda (Sf) cell system has become popular for the production of large amounts of the human erythrocyte glucose transporter, GLUT1, heterologously. However, it was not possible to show that the expressed transporter in insect cells could actually transport glucose. The possible reason for this was that the activity of the endogenous insect glucose transporter was extremely high and so rendered transport activity resulting from the expression of exogenous transporter very difficult to detect. Sf21-AE cells are commonly employed as the host permissive cell line to support the baculovirus AcNPV replication and protein synthesis. The cells grow well on TC-100 medium that contains 0.1 % D-glucose as the major carbon source, strongly suggesting the presence of endogenous glucose transporters. However, unlike the human glucose transporter, very little is known about properties of the endogenous sugar transporter(s) in insect cells. Thus, the uptake of 2-deoxy-D-glucose (2dGlc) by Sf21-AE cells and the inhibition of 2dGlc transport in the insect cells by fructose and cytochalasin B were investigated in the present work. The binding assay of cytochalasin B was also performed, which could be used as a functional assay for the endogenous glucose transporter(s) in the insect cells. Sf21-AE cells were infected with the recombinant virus AcNPV-GT or no virus, at a multiplicity of infection (MOI) of 5. Infected cells were resuspended in PBS plus and minus 300 mM fructose, and plus and minus 20 $\mu$M cytochalasin B for use in transport assays. Uptake was measured at 28$^{\circ}C$ for 1 min, with final concentration of 1 mM deoxy-D-glucose, 2-[1,2-$^3$H]- or glucose, L-[l,$^3$H]-, used at a specific radioactivity of 4 Ci/mol. The results obtained demonstrated that the sugar uptake in uninfected cells was stereospecific, and was strongly inhibited by fructose but only poorly inhibitable by cytochalasin B. It is therefore suggested that the Sf21-AE glucose transporter has very low affinity for cytochalasin B, a potent inhibitor of human erythrocyte glucose transporter.

• Diabetes and Metabolism Journal
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• v.42 no.6
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• pp.465-471
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• 2018

My professional journey to understand the glucose homeostasis began in the 1990s, starting from cloning of the promoter region of glucose transporter type 2 (GLUT2) gene that led us to establish research foundation of my group. When I was a graduate student, I simply thought that hyperglycemia, a typical clinical manifestation of type 2 diabetes mellitus (T2DM), could be caused by a defect in the glucose transport system in the body. Thus, if a molecular mechanism controlling glucose transport system could be understood, treatment of T2DM could be possible. In the early 70s, hyperglycemia was thought to develop primarily due to a defect in the muscle and adipose tissue; thus, muscle/adipose tissue type glucose transporter (GLUT4) became a major research interest in the diabetology. However, glucose utilization occurs not only in muscle/adipose tissue but also in liver and brain. Thus, I was interested in the hepatic glucose transport system, where glucose storage and release are the most actively occurring.

• Journal of Life Science
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• v.4 no.4
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• pp.170-175
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• 1994

The glucose transport across the mammalian plasma membranes is carried out by members of two distinct gene families, $Na^+$/glucose to transporter (SGLT) and glucose transporters (GLUTs). The energy requiring SGLT utilizes the sodium gradient to transport glucose and galactose against the concentration gradient. The energy independent transport (Facilitative transport) of glucose down the concentration gradient is mediated by the members of GLUTs. The facilitated transport of glucose is saturable, sterospecific and bidirectional across the membrane. To date, 6 kinds of isoforms of facilitative glucose transporters are found. These proteins are expressed in a tissue and cell specific manner, and shows distinct properties that reflect their specific functional roles.

• Biomedical Science Letters
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• v.11 no.4
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• pp.487-492
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• 2005

The baculovirus/insect cell expression system is of great value in the study of structure-function relationships in mammalian glucose-transport proteins by site-directed mutagenesis and for the large-scale production of these proteins for mechanistic and biochemical studies. Spodoptera frugiperda Clone 21 (Sf2l) cells grow well on TC-100 medium that contains $0.1\%$ D-glucose as the major carbon source, strongly suggesting the presence of endogenous glucose transporters. However, very little is known about the properties of the endogenous sugar transporter(s) in Sf2l cells, although a saturable transport system for hexose uptake has been previously revealed in the Sf cells. In order to further examine the substrate and inhibitor recognition properties of the Sf2l cell transporter, the ability of hexoses to inhibit 2-deoxy-D-glucose (2dGlc) transport was investigated by measuring inhibition constants $(K_i)$. The $K_i's$ for reversible inhibitors were determined from plots of uptake versus inhibitor concentration. Transport was effectively inhibited by D-mannose and D-glucose. Of the hexoses tested, L-glucose had the least effect on 2dGlc transport in the Sf2l cells, indicating that the transport is stereoselective. Unlike the human HepG2 type glucose transport system, D-mannose had a somewhat greater affinity for the Sf2l cell transporter than D-glucose, implying that the hydroxyl group at the C-2 position is not necessary for strong binding. However, epimerization at the C-4 position of D-glucose (D-galactose) resulted in a dramatic decrease in affinity of the hexose for the Sf2l cell transporter. Such a lowering of affinity might be the result of the involvement of the C-4 hydroxyl in hydrogen bonding. It is therefore suggested that Sf2l cells were found to contain an endogenous sugar transport activity that in several aspects resembles the human HepG2 type glucose transporter, although the insect and human transporters do differ in their affinity for cytochalasin B.

• The Korean Journal of Physiology
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• v.25 no.2
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• pp.115-123
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• 1991

Molecular association of glucose transporters with the other proteins in the plasma membrane was assessed by gel electrophoresis and immunoblot techniques. Approximately $31.5{\pm}5.1%$ of GLUT-4, $64.8{\pm}2.7%$ of clathrin, 48.7% of total protein in the plasma membrane (PM) were found insoluble upon extraction with 1% Tx-100. Sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed that the Tx-100 insoluble PM fraction contained about 4 major polypeptides with apparent molecular weight of above 200, 100-120, 80 and 30-35 KDa that were readily removed upon wash with a high pH buffer which is known to remove clathrin and 0.5 M Tris-buffer which is known to remove assembly proteins (AP). Immunoblotting of GLUT4 and clathrin against specific antibodies showed that GLUT-4 and clathrin were co-solubilized up to 84.6% and 82.7% respectively by wash with a high pH buffer and 1% Tx-100. When the membrane was pre-washed with a high pH buffer and 0.5 M Tris solution, GLUT4 and clathrin were not solubilized further suggesting that GLUT4 molecules are in molecular association with clathrin, AP and/or other extrinsic membrane proteins in plasma membrane and the formation of clathrin-coated structures might be involved in insulin stimulated glucose transporter translocation mechanism.

• Biomedical Science Letters
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• v.7 no.4
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• pp.161-166
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• 2001

Most mammalian cells take up glucose by passive transport proteins in the plasma membranes. The best known of these proteins is the human erythrocyte glucose transporter, GLUT1. High levels of heterologous expression far the transporter are necessary for the investigation of its three-dimensional structure by crystallization. To achieve this, the baculovirus expression system has become popular choice. However, Spodoptera frugiperda Clone 9 (Sf9) cells, which are commonly employed as the host permissive cell line to support baculovirus replication and protein synthesis, grow well on TC-100 medium that contains 0.1% D-glucose as the major carbon source, suggesting the presence of endogenous glucose transporters. Furthermore, very little is known of the endogenous transporters properties of Sf9 cells. Therefore, human GLUT1 antibodies would play an important role for characterization of the GLUT1 expressed in insect cell. However, the successful use of such antibodies for characterization of GLUT1 expression m insect cells relies upon their specificity for the human protein and lack of cross-reaction with endogenous transporters. It is therefore important to determine the potential cross-reactivity of the antibodies with the endogenous insect cell glucose transporters. In the present study, the potential cross-reactivity of the human GLUT1 antibodies with the endogenous insect cell glucose transporters was examined by Western blotting. Neither the antibodies against intact GLUT1 nor those against the C-terminus labelled any band migrating in the region expected fur a protein of M$_r$ comparable to GLUT1, whereas these antibodies specifically recognized the human GLUT1. Specificity of the human GLUT1 antibodies tested was also shown by cross-reaction with the GLUT1 expressed in insect cells. In addition, the insect cell glucose transporter was found to have very low affinity for cytochalasin B, a potent inhibitor of human erythrocyte glucose transporter.

• Clinical and Experimental Reproductive Medicine
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• v.25 no.1
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• pp.77-86
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• 1998

The uptake of glucose for metabolism and growth is essential to most animal cells and is mediated by glucose-transporter (GLUT) proteins. The aim of this study was to determine which class of glucose transporter molecules was responsible for uptake of glucose in the mouse early embryo and at which stage the corresponding genes were expressed. In addition, co-culture system with vero cell was used to investigate the effect of the system on GLUT expression. Two-cell stage embryos were collected from the superovulated ICR female and divided into 3 groups. As a control, embryos were cultured in 0.4% BSA-T6 medium which includes glucose. For the experimental groups, embryos were cultured in either co-culture system with vero cells or glucose-free T6 medium supplemented with 0.4% BSA and pyruvate as an energy substrate. 2-cell to blastocyst stage embryos in those groups were respectively collected into microtubes (50 embryos/tube). Total RNA was extracted and RT-PCR was performed. The products were analysed after staining ethidium bromide by 2% agarose gel electrophoresis. Blastocysts were collected from each group at l20hr after hCG injection. They were fixed in 2.5% glutaraldehyde, stained with hoechst, and mounted for observation. In control, GLUT1 was expressed from 4-cell to blastocyst. GLUT2 and GLUT3 were expressed in morula and blastocyst. GLUT4 was expressed in all stages. When embryos were cultured in glucose-free medium, no significant difference was shown in the expression of GLUT1, 2 and 3, compared to control. However GLUT4 was not expressed until morular stage. When embryos were co-cultured with vero cell, there was no significant difference in the expression of GLUT1, 2, 3 and 4 compared to control. To determine cell growth of embryos, the average cell number of blastocyst was counted. The cell number of co-culture ($93.8{\pm}3.1$, n=35) is significantly higher than that of control and glucose-free group ($76.6{\pm}3.8$, n=35 and $68.2{\pm}4.3$, n=30). This study shows that the GLUT genes are expressed differently according to embryo stage. GLUTs were detectable throughout mouse preimplantation development in control and co-culture groups. However, GLUT4 was not detected from 2- to 8-cell stage but detected from morula stage in glucose-free medium, suggested that GLUT genes are expressed autocrinally in the embryo regardless of the presence of glucose as an energy substrate. In addition, co-culture system can increase the cell count of blastocyst but not improve the expression of GLUT. In conclusion, expression of GLUT is dependent on embryo stage in preimplantation embryo development.