HepG2 Glucose Transporter Research Articles

Amino acid substitutions at tryptophan 388 and tryptophan 412 of the HepG2 (Glut1) glucose transporter inhibit transport activity and targeting to the plasma membrane in Xenopus oocytes.

All 6 tryptophan residues in the human HepG2-type glucose transporter (Glut1) were individually altered by site-directed mutagenesis to investigate the role of these residues in transport function. Tryptophan residues in positions 48, 65, 186, 363, 388, and 412 of Glut1 were changed to either a glycine or leucine residue. Mutant mRNAs were synthesized and injected into Xenopus laevis oocytes. Transporter function as assessed by uptake of 2-deoxy-D-[3H]glucose or transport of 3-O-[3H]methylglucose was decreased in the 388 and 412 mutants but was unaltered in all other mutants. The amount of the mutant transporters expressed in total membrane and plasma membrane fractions was measured using Glut1-specific antibodies. Calculation of the intrinsic transport activity of each of the mutants using these data demonstrated that the reduced transport activity of the 412 mutants was caused entirely by a dramatic decrease in the intrinsic activity of the mutant proteins whereas the reduced activity of the 388 mutants was a result of a decreased level of the protein in oocytes, decreased targeting to the plasma membrane, and a modest decrease in the intrinsic activity. Protease/glycosidase mapping of in vitro translation products indicated that the effects of the 388 and 412 point mutations could not be attributed to a disruption in the ability of the mutant proteins to insert properly into the membrane. The ID50 for cytochalasin B inhibition of 2-deoxyglucose uptake was increased from 5 x 10(-7) M for the wild-type Glut1 to 4 x 10(-6) M in the 388 mutants but was unaltered in the 412 mutants. These observations suggest that 1) Trp-412 may comprise part of a hexose binding site or is involved in maintaining a local tertiary structure critical for transport function; 2) Trp-388 is involved in stabilizing the equilibrium binding of cytochalasin B to the transporter. Trp-388 may therefore lie near a substrate binding site and also appears to participate in stabilization of local tertiary structure important for full catalytic activity and efficient targeting to the Xenopus plasma membrane.

Active and monomeric human red cell glucose transporter after high performance molecular-sieve chromatography in the presence of octyl glucoside and phosphatidylserine or phosphatidylcholine

The human red cell glucose transporter (Glut 1) was purified by ion-exchange chromatography in the presence of octyl glucoside. The state of association of the protein was studied, and the transport activity was determined after exchange of copurified membrane lipids for phosphatidylserine (PS) or phosphatidylcholine (PC). The purpose was to analyze the Glut 1 preparation for homogeneity and activity prior to attempts at crystallization. Analyses by high performance molecular-sieve chromatography showed that the Glut 1 was monomeric immediately after the ion-exchange purification: the M r of the Glut 1 polypeptide was estimated to be 49 000 ± 6000 by TSKgel G3000SW chromatography monitored by low-angle laser light-scattering photometry, differential refractometry and UV photometry. This required determination of the absorption coefficient of the Glut 1, which was measured to be 1.13 ± 0.03ml mg −1 cm −1 at 280 nm, referring to the polypeptide concentration. The M r value is consistent with the cDNA-deduced M r 54117 of the very similar HepG2 glucose transporter polypeptide. At 2°C, pH 7 and an ionic strength of 0.06 M, the Glut 1 associated gradually during three days to form oligomers. These formed much more rapidly at room temperature or at high ionic strength. Freshly prepared Glut 1 retained high activity after separation from membrane lipids on a TSKgel G3000SW column in the presence of 40 mM octyl glucoside and 1 mM PS or PC. In contrast, most of the activity was lost when the membrane lipids were separated from the protein in the absence of eluent lipids. The presence of a phospholipid was thus essential for retention of high activity of the Glut 1 in octyl glucoside and PC was nearly as effective as PS.

Regulation of glucose transporter synthesis in cultured human skin fibroblasts

The present study was designed to see the effects of glucose on glucose transporter expression and glucose transport activity using cultured human skin fibroblasts. When the cells were incubated with various concentrations of glucose (11.1–44.4 mM), no differences were found in the HepG2 glucose transporter mRNA, protein levels and basal and insulin-stimulated 2-deoxyglucose uptake. Glucose deprivation, however, resulted in approximately 4-fold increases in the mRNA and 3-fold increases in the protein and the basal 2-deoxyglucose uptake. Chronic exposure to insulin increased the glucose transporter protein levels to similar degrees in the cells incubated with 11.1, 22.2 and 44.4 mM glucose accompanied by increases in the glucose transport activity. Effects of insulin on the glucose transporter mRNA and protein levels, however, were not evident in the glucose-deprived cells. It is concluded that glucose transport activity correlates closely with HepG2 glucose transporter expression in cultured human fibroblasts and that glucose (11.1–44.4 mM) does not affect the glucose transporter expression and glucose transport activity.

Localization of Na(+)-dependent active type and erythrocyte/HepG2-type glucose transporters in rat kidney: immunofluorescence and immunogold study.

Glucose is actively taken up from the glomerular filtrate into the tubule cells by the Na(+)-dependent active glucose transporter (GT), and passively crosses the basolateral membrane via facilitated diffusion GT. With the use of antibodies directed against two types of GTs, we show the immunocytochemical localization of the Na(+)-dependent active GT (SGLT1) and the erythrocyte/HepG2-type facilitated diffusion GT (GLUT1). For light microscopic observation, frozen sections were stained by the rhodamine labeling method. Counterstaining with fluorescein-phalloidin and 4,6-diamidino-2-phenylindole dihydrochloride (DAPI) was employed to facilitate cell type identification. Immunogold staining was carried out on ultra-thin frozen sections for electron microscopy. The antibody to SGLT1 reacted with a 77 KD protein in immunoblotting of a kidney lysate. By immunocytochemistry, SGLT1 was localized in the microvillous plasma membrane in the apical brush borders of the cells of all three proximal tubule segments (S1, S2, and S3). The antibodies to GLUT1, a member of the facilitated diffusion GT family, were raised against human erythrocyte GT or synthetic oligopeptides derived from HepG2 GT, which reacted with a 48 KD protein in immunoblotting of the kidney lysate. GLUT1 was found at the basolateral plasma membranes of S3 proximal tubule cells, cells of the thick limb of Henle's loop, and collecting duct cells. Combined with known physiological data, our findings suggest that SGLT1 in the apical plasma membrane of the proximal tubule cells is responsible for the Na(+)-dependent active reabsorption of glucose from the glomerular filtrate. GLUT1 in the basolateral plasma membrane of S3 cells may transport reabsorbed glucose to the blood vessels. GLUT1 in the basolateral plasma membranes of cells of the thick limb of Henle's loop and of the collecting duct, on the other hand, may nourish these metabolically active cells by facilitating the diffusion of extracellular glucose provided from blood through the basolateral side of the cells.

Effects of Exercise Training and Dietary Manipulation on Insulin-Regulatable Glucose-Transporter mRNA in Rat Muscle

Both exercise training and dietary manipulation (increasing omega-3/omega-6 fat ratio) can ameliorate insulin resistance caused by a high-fat diet in rats. We determined whether alterations in the expression of the insulin-regulatable (IR) and/or HepG2 glucose-transporter (GT) mRNAs were similarly affected. There was a significantly higher level of IRGT mRNA in skeletal muscle from exercise-trained versus sedentary high-fat-fed rats (27% increase, P less than 0.01). This difference is consistent with previously reported increases in muscle insulin-mediated glucose uptake. Skeletal muscle HepG2GT mRNA was too low to detect any training effect, but there was a tendency toward higher levels with training in cardiac muscle. In contrast, dietary manipulation, previously shown to lead to a much greater increase (100-300%) in muscle insulin-mediated glucose uptake, did not change IRGT or HepG2GT mRNA in skeletal muscle or heart. Thus, both dietary manipulation and exercise training increase insulin-stimulated glucose uptake in skeletal muscle, but only exercise training increases IRGT mRNA. Therefore, exercise training apparently increases GT production, whereas dietary manipulation improves glucose transport in skeletal muscle by other mechanisms.

Localization of the forskolin photolabelling site within the monosaccharide transporter of human erythrocytes

Chemical and proteolytic digestion of intact erythrocyte glucose transporter as well as purified transporter protein has been used to localize the derivatization site for the photoaffinity agent 3-[125I]iodo-4-azido-phenethylamino-7-O-succinyldeacetylforskol in [( 125I]IAPS-forskolin). Comparison of the partial amino acid sequence of the labelled 18 kDa tryptic fragment with the known amino acid sequence for the HepG2 glucose transporter confirmed that the binding site for IAPS-forskolin is between the amino acid residues Glu254 and Tyr456. Digestion of intact glucose transporter with Pronase suggests that this site is within the membrane bilayer. Digestion of labelled transporter with CNBr generated a major radiolabelled fragment of Mr approximately 5800 putatively identified as residues 365-420. Isoelectric focusing of Staphylococcus aureus V8 proteinase-treated purified labelled tryptic fragment identified two peptides which likely correspond to amino acid residues 360-380 and 381-393. The common region for these radiolabelled peptides is the tenth putative transmembrane helix of the erythrocyte glucose transporter, comprising amino acid residues 369-389. Additional support for this conclusion comes from studies in which [125I]APS-forskolin was photoincorporated into the L-arabinose/H(+)-transport protein of Escherichia coli. Labelling of this transport protein was protected by both cytochalasin B and D-glucose. The region of the erythrocyte glucose transporter thought to be derivatized with IAPS-forskolin contains a tryptophan residue (Trp388) that is conserved in the sequence of the E. coli arabinose-transport protein.

Insulin regulation of hexose transport in mouse 3T3-L1 cells expressing the human HepG2 glucose transporter

Complementary DNA encoding a HepG2 cell-facilitated glucose transporter (GLUT1) was subcloned into a metal-inducible, mammalian expression vector, pLEN. Mouse 3T3-L1 fibroblasts transfected with this new construct, pLENGT, exhibited zinc-inducible expression of human glucose transporter mRNA, protein, and glucose transport activity, before and after differentiation into adipocytes. Both mouse host GLUT1 and expressed human GLUT1 proteins distributed about equally between 3T3-L1 adipocyte plasma membranes and low density microsomal membranes, while host skeletal muscle/adipocyte-type glucose transporter (GLUT4) was concentrated in the latter fraction. Mouse GLUT1 and GLUT4 proteins and the constitutively expressed human GLUT1 protein in pLENGT adipocytes were all redistributed from low density microsomal membrane to plasma membrane fractions in response to insulin. Insulin stimulated 2-deoxyglucose uptake in untransfected fibroblasts about 2-fold, while untransfected adipocytes displayed a 14-fold increase in deoxyglucose uptake in response to insulin. Both the expression of human GLUT1 protein and basal 2-deoxyglucose uptake by 75 microM zinc-treated pLENGT fibroblasts and adipocytes were increased approximately 3-fold over untransfected cells. In such pLENGT fibroblasts expressing human GLUT1 protein, however, the absolute values for insulin-stimulated increases in sugar uptake were no different than in control fibroblasts. As was observed in pLENGT fibroblasts, the increased basal sugar uptake by pLENGT adipocytes was additive with the insulin-stimulated increase in the rate of sugar uptake and, therefore, the -fold stimulation by insulin was markedly reduced. These data indicate that: 1) the membrane distributions of a glucose transporter protein, which is not responsive to insulin in HepG2 cells, and both mouse GLUT1 and GLUT4 glucose transporter isoforms are regulated by insulin in mouse 3T3-L1 adipocytes, and 2) the expressed human GLUT1 appears to contribute significantly to the rate of basal uptake but not to the insulin-stimulated increase in 2-deoxyglucose uptake by 3T3-L1 fibroblasts and adipocytes.

Primary structure, genomic organization and heterologous expression of a glucose transporter from Arabidopsis thaliana.

Both genomic and full length cDNA clones of an Arabidopsis thaliana sugar carrier, STP1, have been obtained using a cDNA clone of the H+/hexose cotransporter from the green alga Chlorella kessleri as hybridization probe. The peptide predicted from these sequences in 522 amino acids long and has a molecular weight of 57,518 kd. This higher plant sugar carrier contains 12 putative transmembrane segments and is highly homologous to the H+/hexose cotransporter from Chlorella, with an overall identity in the amino acid sequence of 47.1%. It is also homologous to the human HepG2 glucose transporter (28.4%), and other sugar carriers from man, rat, yeast and Escherichia coli. The definite proof for the function of the STP1 protein as a hexose transporter and data on its substrate specificity were obtained by heterologous expression in the fission yeast Schizosaccharomyces pombe. Transformed yeast cells transport D-glucose with a 100-fold lower KM value than control cells. Moreover only the transformed cells were able to accumulate the non-metabolizable D-glucose analogue 3-O-methyl-D-glucose, indicating that the Arabidopsis carrier catalyses an energy dependent, active uptake of hexoses. Expression of STP1 mRNA is low in heterotrophic tissues like roots or flowers. High levels of expression are found in leaves.

Hep-G2 glucose transporter gene polymorphism in Caucasian, Black, Hispanic and Japanese patients with NIDDM

DNA from non-diabetic Caucasians ( n = 16), Blacks ( n = 22), Hispanics ( n = 13) and Japanese ( n = 21), as well as DNA from 34 Caucasian, 19 Black, 19 Hispanic and 20 Japanese non-insulin-dependent diabetes mellitus (NIDDM) patients were examined for restriction fragment length polymorphism (RFLP) after digestion with enzymes BglII and XbaI, and hybridization with the glucose transporter probe, hGT2-2. There were significant differences in the incidence of the RFLPs between Caucasians and Blacks, both controls and patients with NIDDM. Digestion with XbaI revealed a higher incidence of the homozygotic state for allele I in NIDDM Caucasians (12 vs. 0%) than in controls. In NIDDM Blacks and Hispanics, we found a high incidence of a combination of two traits: 42% of the Black and 47% of the Hispanic NIDDM patients were homozygous for the BglII allele I and heterozygous for XbaI. Only 23% of non-NIDDM Blacks or Hispanics had this combination ( P < 0.05). There was no association between RFLP frequency and NIDDM among Japanese subjects. These data support the influence of race on both BglII and XbaI RFLPs. The homozygotes for XbaI in Caucasians and the presence of two specific traits in Blacks and Hispanics appear with higher frequency in NIDDM.

Insulin action on activity and cell surface disposition of human HepG2 glucose transporters expressed in Chinese hamster ovary cells.

Complementary DNA encoding a facilitative glucose transporter was isolated from a human hepatoma cell line (HepG2) cDNA library and subcloned into a metal-inducible mammalian expression vector, pLEN (California Biotechnology) containing human metallothionein gene II promoter sequences. Chinese hamster ovary (CHO) cells transfected with this transporter expression vector, pLENGT, exhibited a 2-17-fold increase in immunoreactive HepG2-type glucose transporter protein, as measured by protein immunoblotting with antipeptide antibodies directed against the HepG2-type glucose transporter C-terminal domain. Expression of the human glucose transporter was verified by protein immunoblotting with a mouse polyclonal antiserum that recognizes the human but not the rodent HepG2-type transporter. 2-Deoxy-D-glucose uptake was increased 2-7-fold in transfected cell lines. Polyclonal antisera directed against purified red blood cell glucose transporter were raised in several rabbits. Antiserum from one rabbit, delta, was found to bind to the surface of intact red cells but not to inside-out red cell ghosts. Using this delta-antiserum in intact cell-binding assays, 1.6-9-fold increases in cell surface expression of the human glucose transporter were measured in CHO-K1 cell lines transfected with the transporter expression vector. Measurements of total cellular glucose transporter immunoreactive protein using anti-HepG2 transporter C-terminal peptide serum, cell surface glucose transporter protein using delta-antiserum and 2-deoxyglucose uptake revealed proportional relationships among these parameters in transfected cell lines expressing different levels of transporter protein. Insulin increased 2-deoxyglucose uptake 40% in control CHO-K1 cells and in CHO-K1 cells expressing modest levels of the human glucose transporter protein. However, stimulation of sugar-uptake by insulin was only 10% in cells overexpressing human glucose transporter protein 9-fold, and no effect of insulin on sugar uptake was detected in several cell lines expressing very high levels (12-17-fold over controls) of human HepG2 glucose transporter protein. No insulin stimulation of anti-cell surface glucose transporter antibody binding was detected in any control or transfected CHO-K1 cell lines. These data indicate that a glucose transporter protein that is insensitive to insulin in HepG2 cells is regulated by insulin when expressed at low but not at high levels in insulin-response CHO-K1 cells. Additionally, the results suggest that insulin does not increase 2-deoxyglucose uptake by increasing the number of cell surface HepG2-type glucose transporters in CHO-K1 fibroblasts.

Restriction Site Polymorphisms at the Human HepG2 Glucose Transporter Gene Locus in Caucasian and West Indian Subjects with Non-Insulin-Dependent Diabetes mellitus

Digestion of human genomic DNA with the restriction enzyme StuI revealed a 2-allele polymorphism with a human HepG2 glucose transporter probe. Bands of 3.2 kilobases (kb; S1 allele) and 2.6 kb (S2 allele) were observed. The genotype frequencies were investigated in 2 non-insulin-dependent diabetic populations. The genotype frequencies of S1S1, S1S2 and S2S2 were 6, 42 and 52% among Caucasian diabetic subjects (n = 48), and 11, 38 and 51% in 47 controls, respectively. In West Indian diabetic patients (n = 48), the genotype frequencies were 17, 54 and 29%, and for 36 controls they were 25, 33 and 42%, respectively. The polymorphism information content of this restriction fragment length polymorphism (RFLP) is 0.32 in Caucasians and 0.37 in West Indians, respectively. There was no significant difference of allele or genotype frequencies between the diabetic patients and non-diabetic controls in either group. Haplotype analysis of the StuI and XbaI RFLPs showed that there was also no significant difference in the frequencies of the four different haplotypes S1X1, S1X2, S2X1 and S2X2 between the patients and controls. However, there was a difference for the frequency of the S1 allele between Caucasians (controls 30%, patients 27%) and West Indians (controls 42%, patients 44%). There was also a significant difference in the frequency of haplotype S2X2 between these two racial groups (controls 48%, cases 51% for Caucasians, and controls 33%, cases 22% for West Indians).

Functional expression of the human HepG2 and rat adipocyte glucose transporters in Xenopus oocytes: Comparison of kinetic parameters

Facilitated glucose transport is a ubiquitous characteristic of animal cells carried out by a family of membrane glycoproteins. Two members of this gene family are the well characterized human erythrocyte protein that has been cloned from the HepG2 cell line and the insulin-sensitive transporter that has been cloned from adipocytes and muscle tissue. In the present study the HepG2 and adipocyte glucose transporters were functionally expressed in Xenopus oocytes after injection of synthetic mRNAs produced by transcription in vitro from cloned cDNAs. Both 2-deoxyglucose uptake and 3-O-methylglucose transport were increased several-fold over basal levels in mRNA-injected oocytes. Increased uptake of 2-deoxyglucose was inhibited completely in the presence of cytochalasin B, and 3-O-methylglucose transport was blocked by D-glucose but not by L-glucose. The half-saturation constant and turnover number for 3-O-methylglucose transport at 22 degrees C via the HepG2 transporter were estimated to be 21 mM and 2.2 x 10(3) s-1 under equilibrium exchange conditions. The half-saturation constant for 3-O-methylglucose transport via the adipocyte transporter under the same conditions was estimated to be 1.8 mM. These data prove the functional identity of the cloned HepG2 and adipocyte cDNAs and indicate that the HepG2 and adipocyte transporters display similar kinetic behavior when expressed in the frog oocyte membrane as compared with their native membrane environments. Thus, the difference in the equilibrium exchange kinetic parameters for glucose transport in the erythrocyte and the adipocyte is a result of the expression of two distinct glucose transporter proteins.

Monokine regulation of glucose transporter mRNA in L6 myotubes

Endotoxin-induced macrophage secretory proteins (monokines) have been shown to stimulate hexose uptake in L6 myotubes (1). In those studies a doubling of the Vmax for hexose uptake was observed which correlated with elevated numbers of glucose transporters (GT) in both plasma and microsomal membranes. To determine if these changes in transporter populations were due to increased GT mRNA, we performed Northern blot analysis using L6 cell RNA and a cDNA to the HepG2 glucose transporter. The L6 myotubes contained a single 2.8 kb species of GT mRNA that increased 2.5-fold after an 8h exposure to the monokine preparation. β-Actin mRNA levels were unaltered by the treatment ,indicating specificity of monokine action. Glucose transporter mRNA content appeared to reach a maximum 8h after exposure to the monokine. Over the next 16h the levels of this mRNA gradually decreased, approaching control levels. Data obtained from nuclear transcription run-on assays suggest that increased levels of GT mRNA are due to an increased rate of gene transcription. A second transporter, the insulin-sensitive glucose transporter, was also observed to be expressed in the L6 cells. Monokine treatment resulted in a 60% suppression of the mRNA coding for this protein.

Differential regulation of two glucose transporters in adipose cells from diabetic and insulin-treated diabetic rats.

At least two genetically distinct glucose transporters (GTs) coexist in adipose cells, one cloned from human hepatoma cells and rat brain (HepG2/brain) and another from rat skeletal muscle, heart, and adipose cells (adipose cell/muscle). Here we demonstrate differential regulation of these two GTs in adipose cells of diabetic and insulin-treated diabetic rats and compare changes in the expression of each GT with marked alterations in insulin-stimulated glucose transport activity. Adipose cell/muscle GTs detected by immunoblotting with the monoclonal antiserum 1F8 (James, D. E., R. Brown, J. Navarro, and P. F. Pilch. 1988. Nature (Lond.). 333:183-185), which reacts with the protein product of the newly cloned adipose cell/muscle GT cDNA, decrease 87% with diabetes and increase to 8.5-fold diabetic levels with insulin treatment. These changes concur qualitatively with previous detection of GTs by cytochalasin B binding and with insulin-stimulated 3-O-methylglucose transport. Northern blotting reveals that the adipose/muscle GT mRNA decreases 50% with diabetes and increases to 6.8-fold control (13-fold diabetic) levels with insulin treatment. In contrast, GTs detected with antisera to the carboxyl terminus of the HepG2 GT or to the human erythrocyte GT show no significant change with diabetes or insulin treatment. The HepG2/brain GT mRNA is unchanged with diabetes and increases threefold with insulin treatment. These results suggest that (a) altered expression of the adipose cell/muscle GT forms the molecular basis for the dysregulated glucose transport response to insulin characteristic of diabetes, (b) the expression of two types of GTs in rat adipose cells is regulated independently, and (c) alterations in mRNA levels are only part of the mechanism for in vivo regulation of the expression of either GT species.

Coordinate regulation of glucose transporter function, number, and gene expression by insulin and sulfonylureas in L6 rat skeletal muscle cells.

The extrapancreatic actions of sulfonylureas on the glucose transport system were studied in the L6 line of cultured rat skeletal muscle cells. Insulin (10(-7) M) increased 2-deoxyglucose uptake in differentiated L6 myotubes by 30-40% after 8 h of incubation. The sulfonylurea tolazamide (0.6 mg/ml, 22 h) had no effect on glucose uptake in the absence of insulin, but increased insulin-stimulated 2-deoxyglucose uptake twofold. The total cellular content of glucose transporters was assessed with a monoclonal anti-transporter antibody by a solid-phase ELISA method. Insulin (8 h) increased the quantity of glucose transporters, with a maximal twofold increase at 10(-7) M and a dose-response curve similar to that for insulin stimulation of glucose uptake. In spite of its lack of effect on glucose uptake, tolazamide alone (0.6 mg/ml) increased the cellular content of transporters by 70%. The effects of insulin and tolazamide on transporter gene expression were studied with probes derived from Hep G2 glucose transporter cDNA. Insulin increased the transporter mRNA level 1.7-fold, tolazamide increased it 1.5-fold, and the combination of insulin and tolazamide increased transporter mRNA 3-fold. It is concluded that sulfonylureas, together with insulin, enhance glucose uptake in L6 skeletal muscle cells by increasing the number of functioning glucose transport molecules. The long-term regulation of the glucose transport system in skeletal muscle by insulin and sulfonylureas in vivo may involve similar changes in transporter function, number, and gene expression.

Molecular cloning and characterization of an insulin-regulatable glucose transporter.

A major mechanism by which insulin stimulates glucose transport in muscle and fat is the translocation of glucose transporters from an intracellular membrane pool to the cell surface. The existence of a distinct insulin-regulatable glucose transporter was suggested by the poor cross-reactivity between antibodies specific for either the HepG2 or rat brain glucose transporters and the rat adipocyte glucose transporter. More direct evidence was provided by the production of a monoclonal antibody (mAb 1F8) specific for the rat adipocyte glucose transporter that immunolabels a species of relative molecular mass 43,000 (43K) present only in tissues that exhibit insulin-dependent glucose transport, suggesting that this protein may be encoded by a different gene from the previously described mammalian glucose transporters. This antibody has been used to immunoprecipitate a 43K protein that was photoaffinity-labelled with cytochalasin B in a glucose displaceable way, and to immunolabel a protein in the plasma membrane of rat adipocytes, whose concentration was increased at least fivefold after cellular insulin exposure. Here we describe the cloning and sequencing of cDNAs isolated from both rat adipocyte and heart libraries that encode a protein recognized by mAb 1F8, and which has 65% sequence identity to the human HepG2 glucose transporter. This cDNA hybridizes to an mRNA present only in skeletal muscle, heart and adipose tissue. Our data indicate that this cDNA encodes a membrane protein with the characteristics of the translocatable glucose transporter expressed in insulin-responsive tissues.

Insulin-stimulated Translocation of the HepG2/Erythrocyte-type Glucose Transporter Expressed in 3T3-L1 Adipocytes

Insulin stimulates glucose transport into adipocytes, at least in part, via the translocation of intracellular transporters to the plasma membrane. The human HepG2-type transporter, which is not insulin-responsive in its native cell type, was expressed in 3T3-L1 adipocytes by infection with recombinant retrovirus harboring the HepG2 transporter cDNA in order to determine whether glucose transporter translocation in adipocytes is restricted to a distinct insulin-sensitive transporter species. The distributions of the endogenous murine and the HepG2 transporters were estimated by quantitative immunoblot analysis of subcellular fractions probed with either a monoclonal antibody that recognized only the human transporter or a polyclonal antibody that recognized both transporter species. In the basal state, the intracellular membrane fraction comprised approximately 50% of the total of each transporter type. Insulin decreased the content of both transporter species in the intracellular membranes by approximately 50% and increased the plasma membrane content of both species by approximately 1.5-2-fold. The similar insulin-mediated increase in the plasma membrane content of endogenous murine and HepG2 glucose transporters was verified by labeling of cell surface glycoproteins with [3H]NaBH4 followed by immunoprecipitation with glucose transporter antibodies. These data indicate that insulin-mediated translocation in 3T3-L1 adipocytes is not restricted to a tissue-specific insulin-responsive glucose transporter species and suggest that other tissue-specific factors regulate the translocation process.

Regulation of glucose transporter-specific mRNA levels in rat adipose cells with fasting and refeeding. Implications for in vivo control of glucose transporter number.

Fasting in the rat is associated with a rapid and progressive decrease in insulin-stimulated glucose transport activity in adipose cells, which is not only restored to normal, but increased transiently to supranormal levels by refeeding. The mechanisms for these changes in glucose transport activity appear to involve alterations in both glucose transporter number and intrinsic activity (glucose turnover number). In this study, we use the human hepatoma Hep G2 glucose transporter complementary DNA clone to examine the molecular basis for these alterations. Extractable RNA per adipose cell is decreased 35% with 3 d of fasting and increased to 182% of control with 6 d of refeeding after 2 d of fasting. This parallels changes in adipose cell intracellular water, so that total RNA/water space remains relatively constant. When the changes in total RNA/cell are taken into account, Northern and slot blot analyses with quantitative densitometry reveal a 36% decrease in specific glucose transporter mRNA level in cells from the fasted rats. The mRNA level in cells from the fasted/refed rats is restored to normal. These observations correlate closely with previous measurements of glucose transporter number in adipose cell membrane fractions using cytochalasin B binding and Western blotting. The levels of specific mRNAs for tubulin and actin on a per cell basis show similar but more dramatic changes and mRNAs encoding several differentiation-dependent adipose cell proteins are also significantly affected. Thus, the levels of mRNA for multiple adipose cell genes are affected by fasting and refeeding. In particular, this demonstrates that in vivo metabolic alterations can influence the level of a glucose transporter mRNA in adipose cells. This may have implications for the regulation of glucose transporter number and glucose transport activity.

Regulation of glucose-transporter gene expression by insulin in cultured human fibroblasts.

To clarify the effect of insulin on glucose-transporter (GT) biosynthesis, we determined GT mRNA levels in human cultured skin fibroblasts, using HepG2 GT cDNA as a probe. Insulin specifically increased the GT mRNA level in a time- and dose-dependent manner. Time-course study demonstrated that the mRNA level peaked within 3 h of insulin (1 x 10(-7) M) addition. After remaining elevated for several hours, mRNA decreased and returned to the basal level after 24 h. In the cell strains from seven normal subjects, the mean (+/- SE) GT mRNA level determined after 3 h of treatment with 1 x 10(-7) M insulin was 164.3 +/- 8.5% of the level found in untreated control cells. The insulin dose-response curve of GT mRNA levels showed that the maximum stimulation was elicited at 1 x 10(-7) M, and the half-maximum stimulation occurred at approximately 5 x 10(-10) M. Degradation rates of GT mRNA determined in the presence of actinomycin D were not different between insulin-treated and untreated cells. These results suggest that insulin increases GT gene expression in cultured human fibroblasts.

Studies with antipeptide antibody suggest the presence of at least two types of glucose transporter in rat brain and adipocyte.

Three antipeptide antibodies were prepared by immunizing rabbits with synthesized short peptides corresponding to residues 215-226, 466-479, and 478-492 predicted from the cDNA of both the human hepatoma HepG2 and rat brain glucose transporters. All three antibodies were found to precipitate quantitatively the [3H]cytochalasin B photoaffinity-labeled human erythrocyte glucose transporter. Each antibody also recognized the rat brain protein of Mr 45,000 on immunoblots, and a similar molecular weight protein was labeled with [3H]cytochalasin B in a D-glucose-inhibitable manner, suggesting that this protein is glucose transporter. However, only up to 30% of the labeled rat brain glucose transporters were precipitated, even by repeated rounds of immunoprecipitation. In addition, these antibodies were observed to be unable to immunoprecipitate significantly the [3H]cytochalasin B-labeled rat adipocyte glucose transporter. Further, one-dimensional peptide maps of [3H]cytochalasin B-labeled human erythrocyte and adipocyte glucose transporters generated distinct tryptic fragments. Although Mr 45,000 protein in rat adipocyte low density microsomes was detected on immunoblots and its amount was decreased in insulin-treated cells, the rat adipocyte low density microsomes were much less reactive on immunoblots than the rat brain membranes in spite of the fact that the rat adipocyte low density microsomes contained more [3H]cytochalasin B-labeled glucose transporters. In addition, the ratio of cytochalasin B-labeled glucose transporter per unit HepG2-type glucose transporter mRNA was more than 10-fold higher in rat adipocyte than in rat brain. These results indicate that virtually all the human erythrocyte glucose transporters are of the HepG2 type, whereas this type of glucose transporter constitutes only approximately 30 and 3% of all the glucose transporters present in rat brain and rat adipocyte, respectively; and the rest, of similar molecular weight, is expressed by a different gene.