The Uptake of 2-deoxy-D-glucose (2dGlc) by the Endogenous Sugar Transporter(s) of Spodoptera frugiperda CLone 21-AE Cells and the Inhibition of 2dGlc Transport in the Insect Cells by Fructose and Cytoc halasin B

Chong-Kee Lee 대한의생명과학회 2003 Biomedical Science Letters Vol.9 No.4

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 цM cytochalasin B for use in transport assays. Uptake was measured at 28℃ for 1 min, with final concentration of 1 mM deoxy-D-glucose, 2-[1,2-³H]- or glucose, L-[1, ³H]-, 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.

The Uptake of 2-deoxy-D-glucose (2dGlc) by the Endogenous Sugar Transporter(s) of Spodoptera frugiperda Clone 21-AE Cells and the Inhibition of 2dGIc Transport in the Insect Cells by Fructose and Cytoc halasin B

Lee, Chong-Kee The Korean Society for Biomedical Laboratory Scien 2003 Journal of biomedical laboratory sciences Vol.9 No.4

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.

Investigation of the Nature of the Endogenous Glucose Transporter(s) in Insect Cells

Lee, Chong-Kee 경북대학교 의학연구소 2000 경북대학교병원의학연구소논문집 Vol.4 No.1

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 Km 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-glueose transport was examined by measuring inhibition constants (Ki). 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 tee human erythrocyte glucose transporter. However, the mammalian and insect transporters were different in same of their kinetic properties, namely, their affinities for fructose and for cytochalasin B.

The Effect of Glucose and Glucose Transporter on Regulation of Lactation in Dairy Cow

Heo, Young-Tae,Park, Joung-Jun,Song, Hyuk The Korean Society of Animal Reproduction 2015 Reproductive & developmental biology Vol.39 No.4

Glucose is universal and essential fuel of energy metabolism and in the synthesis pathways of all mammalian cells. Glucose is the one of the major precursors of lactose synthesis using glycolysis result in producing milk fat and protein. During the milk fat synthesis, lipoprotein lipase (LPL) and CD36 are required for glucose uptake. Various morecules such as acyl-CoA synthetase 1 (ACSL1) activity of acetyl-CoA synthetase 2 (ACSS2), ACACA, FASN AGPAT6, GPAM, LPIN1 are closely related with milk fat synthesis. Additionally, glucose plays a major role for synthesizing lactose. Activations of lactose synthesize enzymes such as membranebound enzyme, beta-1,4-galactosyl transferase (B4GALT), glucose-6-phosphate dehydrogenase (G6PD) are changed by concentration of glucose in blood resulting change of amount of lactose production. Glucose transporters are a wide group of membrane proteins that facilitate the transport of glucose over a plasma membrane. There are 2 types of glucose transporters which consisted facilitative glucose transporters (GLUT); and sodium-dependent transport, mediated by the Na+/glucose cotransporters (SGLT). Among them, GLUT1, GLUT8, GLUT12, SGLT1, SGLT2 are main glucose transporters which involved in mammary gland development and milk synthesis. However, more studies are required for revealing clear mechanism and function of other unknown genes and transporters. Therefore, understanding of the mechanisms of glucose usage and its regulation in mammary gland is very essential for enhancing the glucose utilization in the mammary gland and improving dairy productivity and efficiency.

Cross-reactivity of Human Polyclonal Anti-GLUT1 Antisera with the Endogenous Insect Cell Glucose Transporters and the Baculovirus-expressed GLUT1

Lee, Chong-Kee The Korean Society for Biomedical Laboratory Scien 2001 Journal of biomedical laboratory sciences Vol.7 No.4

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.

The Effect of Glucose and Glucose Transporter on Regulation of Lactation in Dairy Cow

Young-Tae Heo,Joung-Jun Park,Hyuk Song 한국동물생명공학회(구 한국동물번식학회) 2015 Reproductive & developmental biology Vol.39 No.4

Glucose is universal and essential fuel of energy metabolism and in the synthesis pathways of all mammalian cells. Glucose is the one of the major precursors of lactose synthesis using glycolysis result in producing milk fat and protein. During the milk fat synthesis, lipoprotein lipase (LPL) and CD36 are required for glucose uptake. Various morecules such as acyl-CoA synthetase 1 (ACSL1) activity of acetyl-CoA synthetase 2 (ACSS2), ACACA, FASN AGPAT6, GPAM, LPIN1 are closely related with milk fat synthesis. Additionally, glucose plays a major role for synthesizing lactose. Activations of lactose synthesize enzymes such as membranebound enzyme, beta-1,4- galactosyl transferase (B4GALT), glucose-6-phosphate dehydrogenase (G6PD) are changed by concentration of glucose in blood resulting change of amount of lactose production. Glucose transporters are a wide group of membrane proteins that facilitate the transport of glucose over a plasma membrane. There are 2 types of glucose transporters which consisted facilitative glucose transporters (GLUT); and sodium-dependent transport, mediated by the Na+/glucose cotransporters (SGLT). Among them, GLUT1, GLUT8, GLUT12, SGLT1, SGLT2 are main glucose transporters which involved in mammary gland development and milk synthesis. However, more studies are required for revealing clear mechanism and function of other unknown genes and transporters. Therefore, understanding of the mechanisms of glucose usage and its regulation in mammary gland is very essential for enhancing the glucose utilization in the mammary gland and improving dairy productivity and efficiency.

The Effect of Glucose and Glucose Transporter on Regulation of Lactation in Dairy Cow

Young-Tae Heo,Joung-Jun Park,Hyuk Song 한국동물번식학회 2015 Reproductive & developmental biology Vol.39 No.4

Glucose is universal and essential fuel of energy metabolism and in the synthesis pathways of all mammalian cells. Glucose is the one of the major precursors of lactose synthesis using glycolysis result in producing milk fat and protein. During the milk fat synthesis, lipoprotein lipase (LPL) and CD36 are required for glucose uptake. Various morecules such as acyl-CoA synthetase 1 (ACSL1) activity of acetyl-CoA synthetase 2 (ACSS2), ACACA, FASN AGPAT6, GPAM, LPIN1 are closely related with milk fat synthesis. Additionally, glucose plays a major role for synthesizing lactose. Activations of lactose synthesize enzymes such as membranebound enzyme, beta-1,4-galactosyl transferase (B4GALT), glucose-6-phosphate dehydrogenase (G6PD) are changed by concentration of glucose in blood resulting change of amount of lactose production. Glucose transporters are a wide group of membrane proteins that facilitate the transport of glucose over a plasma membrane. There are 2 types of glucose transporters which consisted facilitative glucose transporters (GLUT); and sodium-dependent transport, mediated by the Na+/glucose cotransporters (SGLT). Among them, GLUT1, GLUT8, GLUT12, SGLT1, SGLT2 are main glucose transporters which involved in mammary gland development and milk synthesis. However, more studies are required for revealing clear mechanism and function of other unknown genes and transporters. Therefore, understanding of the mechanisms of glucose usage and its regulation in mammary gland is very essential for enhancing the glucose utilization in the mammary gland and improving dairy productivity and efficiency.

복막투석시 포도당의 이동과 한외여과 한외여과에 당 수송체가 미치는 영향

김화정(Hwa Jeong Kim),박민선(Min Sun Park),윤견일(Kun Il Yoon) 대한신장학회 2001 Kidney Research and Clinical Practice Vol.20 No.4

Purpose - The purpose of this study was to evaluate the role of glucose transporter in peritoneal glucose and fluid transport. Methods: Male Sprague-Dawley rats were used. 5mL normal saline with(CB) and without(C) Cytochalasin B(1 ㎛) was intraperitoneally injected once. From the next day 25mL commercial dialysis solutions containing 4.25M glucose was injected into the peritoneal cavity twice a day for 8 weeks in a half of each group(CB-IP, n=6 and C-IP, n=8). The other half of each group served as control without IP(C- Control, n=7 and CB-Control, n=7). A 2 hour dwell study was performed using dialysis solutions containing 4.25Yo glucose. Intraperitoneal volume(IPV) after 2 hours of dwell was measured and peritoneal fluid absorption rate(Qa) was calculated as RISA disappearance rate. Dialysate glucose amount remaining after 2 hour dwell(DGA) was calculated and ex- pressed as 96 of the initial value. Results: IPV was significantly higher in CB than in C in both IP and Control IPV was significantly lower in C-IP than in C-Control and CB-IP while it was similar between CB-Control and CB-IP. Qa was significantly higher in IP than in Control. DGR was significantly higher in CB than in C and in control than in IP. Conclusion: Longterm peritoneal exposure to high glucose dialysis solution increased peritoneal glucose absorption and decreased ultrafiltration volume in rat. A single IP use of glucose transporter inhibitor attenuated increased glucose absorption and decreased ultrafiltration after longterm peritoneal exposure to dialysate.

Investigation of the Nature of the Endogenous Glucose Transporter(s) in Insect Cells

Lee, Chong-Kee Korean Society for Biochemistry and Molecular Biol 1999 Journal of biochemistry and molecular biology Vol.32 No.5

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.

Investigation of the Nature of the Endogenous Glucose Transporter(s) in Insect Cells

Lee, Chong-Kee The Korea Science and Technology Center 1999 BMB Reports Vol.32 No.5

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?? 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??). 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, tha mammalian and insect transporters were different in some of their kinetic properties, namely, their affinities for fructose and for cytochalasin B.