Distinct Glucose Transporters Research Articles

Visualization of multiple localizations of GLUT4 by fluorescent probes of PYP-tag with designed unnatural warhead.

Within a cell, multiple copies of the same protein coexist in different pathways and behave differently. Being able to individually analyze the constant actions of proteins in a cell is crucial to know the pathways through which they pass and which physiological functions they are deeply involved in. However, until now, it has been difficult to distinguish protein copies with distinct translocation properties by fluorescence labeling with different colors in living cells. In this study, we have created an unnatural ligand with an unprecedented protein-tag labeling property in living cells and overcome the above-mentioned problem. Of special interest is that some fluorescent probes with the ligand can selectively and efficiently label intracellular proteins without binding to cell-surface proteins, even if the proteins are present on the cell membrane. We also developed a cell-membrane impermeable fluorescent probe that selectively labels cell-surface proteins without labeling of intracellular proteins. These localization-selective properties enabled us to visually discriminate two kinetically distinct glucose transporter 4 (GLUT4) molecules that show different multiple subcellular localization and translocation dynamics in live cells. Taking advantage of the probes, we revealed that N-glycosylation of GLUT4 influences intracellular localization. Furthermore, we were able to visually distinguish active GLUT4 molecules that underwent membrane translocation at least twice within an hour from those that remained intracellularly, discovering previously unrecognized dynamic behaviors of GLUT4. This technology provides not only a valuable tool for study on multiple localization and dynamics of proteins but also important information on diseases caused by protein translocation dysfunction.

Glucose Transporters and Virulence in Leishmania mexicana.

Glucose transporters are important for viability and infectivity of the disease-causing amastigote stages of Leishmania mexicana The Δgt1-3 null mutant, in which the 3 clustered glucose transporter genes, GT1, GT2, and GT3, have been deleted, is strongly impaired in growth inside macrophages in vitro We have now demonstrated that this null mutant is also impaired in virulence in the BALB/c murine model of infection and forms lesions considerably more slowly than wild-type parasites. Previously, we established that amplification of the PIFTC3 gene, which encodes an intraflagellar transport protein, both facilitated and accompanied the isolation of the original Δgt1-3 null mutant generated in extracellular insect-stage promastigotes. We have now isolated Δgt1-3 null mutants without coamplification of PIFTC3 These amplicon-negative null mutants are further impaired in growth as promastigotes, compared to the previously described null mutants containing the PIFTC3 amplification. In contrast, the GT3 glucose transporter plays an especially important role in promoting amastigote viability. A line that expresses only the single glucose transporter GT3 grows as well inside macrophages and induces lesions in animals as robustly as do wild-type amastigotes, but lines expressing only the GT1 or GT2 transporters replicate poorly in macrophages. Strikingly, GT3 is restricted largely to the endoplasmic reticulum in intracellular amastigotes. This observation raises the possibility that GT3 may play an important role as an intracellular glucose transporter in the infectious stage of the parasite life cycle.IMPORTANCE Glucose transport plays important roles for in vitro growth of insect-stage promastigotes and especially for viability of intramacrophage mammalian host-stage amastigotes of Leishmania mexicana However, the roles of the three distinct glucose transporters, GT1, GT2, and GT3, in parasite viability inside macrophages and virulence in mice have not been fully explored. Parasite lines expressing GT1 or GT2 alone were strongly impaired in growth inside macrophages, but lines expressing GT3 alone infected macrophages and caused lesions in mice as robustly as wild-type parasites. Notably, GT3 localizes to the endoplasmic reticulum of intracellular amastigotes, suggesting a potential role for salvage of glucose from that organelle for viability of infectious amastigotes. This study establishes the unique role of GT3 for parasite survival inside host macrophages and for robust virulence in infected animals.

GLUT12 promotes prostate cancer cell growth and is regulated by androgens and CaMKK2 signaling.

Despite altered metabolism being an accepted hallmark of cancer, it is still not completely understood which signaling pathways regulate these processes. Given the central role of androgen receptor (AR) signaling in prostate cancer, we hypothesized that AR could promote prostate cancer cell growth in part through increasing glucose uptake via the expression of distinct glucose transporters. Here, we determined that AR directly increased the expression of SLC2A12, the gene that encodes the glucose transporter GLUT12. In support of these findings, gene signatures of AR activity correlated with SLC2A12 expression in multiple clinical cohorts. Functionally, GLUT12 was required for maximal androgen-mediated glucose uptake and cell growth in LNCaP and VCaP cells. Knockdown of GLUT12 also decreased the growth of C4-2, 22Rv1 and AR-negative PC-3 cells. This latter observation corresponded with a significant reduction in glucose uptake, indicating that additional signaling mechanisms could augment GLUT12 function in an AR-independent manner. Interestingly, GLUT12 trafficking to the plasma membrane was modulated by calcium/calmodulin-dependent protein kinase kinase 2 (CaMKK2)-5'-AMP-activated protein kinase (AMPK) signaling, a pathway we previously demonstrated to be a downstream effector of AR. Inhibition of CaMKK2-AMPK signaling decreased GLUT12 translocation to the plasma membrane by inhibiting the phosphorylation of TBC1D4, a known regulator of glucose transport. Further, AR increased TBC1D4 expression. Correspondingly, expression of TBC1D4 correlated with AR activity in prostate cancer patient samples. Taken together, these data demonstrate that prostate cancer cells can increase the functional levels of GLUT12 through multiple mechanisms to promote glucose uptake and subsequent cell growth.

Comparison of GLUT1, GLUT2, GLUT4 and SGLT1 mRNA Expression in the Salivary Glands and Six Other Organs of Control, Streptozotocin-Induced and Goto-Kakizaki Diabetic Rats

Background/Aims: The expression and localization of several distinct glucose transporters (GLUT1, GLUT2, GLUT4, and SGLT1) was recently characterized in the parotid gland of normal rats by quantitative real-time PCR analysis, immunohistochemistry and Western blotting. The major aims of the present study was to compare the mRNA expression of these glucose transporters in both the parotid gland and submaxillary gland of control rats, streptozotocin-induced diabetic rats and hereditarily diabetic Goto-Kakizaki rats. Methods: Quantitative real-time PCR analysis was performed in the parotid and submaxillary salivary glands and, for purpose of comparison, also in the heart, kidney, liver, lung, muscle and pancreas from control animals and either streptozotocin-treated or Goto-Kakizaki rats. Results: The expression of GLUT4, but not GLUT1 or SGLT1, mRNA was decreased in the diabetic rats. The results also allow comparing both the mRNA expression level of the four glucose transporters in salivary glands and six other organs, and the diabetes-induced changes in such an expression in distinct locations. Conclusion: The mRNA expression of the insulin-dependent GLUT4 transporter was the sole to be significantly decreased in the salivary glands of diabetic animals. The possible consequence of such a decrease in terms of the control of salivary glucose concentration requires further investigation.

Glucose transporter/T1R3‐expressing cells in rat tracheal epithelium

Glucose transport plays an important role in maintaining low sugar concentration in airway surface liquid (ASL), which is critical for mucociliary clearance and bacterial colonization. Experimental evidence indicates that glucose/hexose uptake in lung/airway cells occurs by means of two structurally distinct glucose transporter pathways: the Na(+) -dependent glucose transporters (SGLT family) and the facilitative glucose transporters (GLUT family). In this study, we examined the expression of the major glucose transporters of the intestine, GLUT2, GLUT5, SGLT1 and T1R3 taste receptor subunit, in the trachea of rats using immunohistochemistry and immunoelectron microscopy, and compared them using double-labeled confocal microscopy. We found that GLUT2, GLUT5, SGLT1 and T1R3 are selectively expressed in different cell types. T1R3 and GLUT2 are predominantly expressed in subsets of solitary chemoreceptor cells (SCCs) and ciliated cells, GLUT5 is present in subsets of SCCs and in secretory cells, and SGLT1 is exclusively expressed in a unique cell type, SCCs. Furthermore, we demonstrated that T1R3 is colocalized with SGLT1 in SCCs and with GLUT2 transporter in ciliated cells. In conclusion, these findings reveal that different cell types are associated with the uptake of glucose in ASL and that, due to their T1R3 expression, SCCs and ciliated cells are most likely to participate in the chemosensory process in ASL.

Differential Glucose Transporter Expression during Perinatal and Postnatal Erythropoiesis

Glucose is a major source of energy for living organisms and its transport in vertebrates is a universally conserved property. Of all cell lineages, human erythrocytes express the highest level of the Glut1 glucose transporter with >200,000 molecules/cell. However, we recently reported that erythrocyte Glut1 expression is a specific trait of vitamin C-deficient mammalian species, comprising only higher primates, guinea pigs and fruit bats (Montel-Hagen et al., Cell, 2008). We now show that in all other tested mammals, including mice, rats, dogs and cows, Glut1 is in fact transiently expressed in erythrocytes during the neonatal period. This is in marked contrast with humans, where Glut1 is present at equivalently high levels on both neonatal and adult RBC. In mice, we found that Glut1 expression was not associated with primitive erythropoiesis but was highly expressed during definitive fetal erythropoiesis. Indeed, this transporter was present at significantly earlier stages of erythropoiesis in fetal spleen and liver than immediately following birth. It was therefore important to determine whether erythrocyte Glut1 expression in mice is specifically associated with fetal erythropoiesis or alternatively, is common to any physiological state where an extensive erythropoiesis is provoked. Induction of a hemolytic anemia in adult mice resulted in a massive erythropoiesis with significant increase in glucose uptake but notably, Glut1 was not detected. Rather, in these conditions as well as following birth, Glut4, an insulin-sensitive transporter previously thought to be responsible for glucose uptake in muscle and adipose tissue, was highly expressed. Following birth, the concomitant repression of Glut1 and induction of Glut4 was associated with a significantly augmented ratio of the Sp3 to Sp1 zinc-finger transcription factors. Thus, in contrast to humans, murine Glut1 is highly expressed during definitive erythropoiesis and is then downregulated at birth. Further erythroid development is characterized by the upregulation of a distinct glucose transporter, Glut4. Expression of distinct glucose transporters in nonhuman erythrocytes, regulated at the transcriptional level, therefore characterizes different states of erythroid development and differentiation.

Differential Regulation of Multiple Glucose Transporter Genes in Leishmania mexicana

We have studied the structure and expression of glucose transporter genes in the parasitic protozoan Leishmania mexicana. Three distinct glucose transporter isoforms, LmGT1, LmGT2, and LmGT3, are encoded by single copy genes that are clustered together at a single locus. Quantitation of Northern blots reveals that LmGT2 mRNA is present at approximately 15-fold higher level in promastigotes, the insect stage of the parasite life cycle, compared with amastigotes, the intracellular stage of the life cycle that lives within the mammalian host. In contrast, LmGT1 and LmGT3 mRNAs are expressed at similar levels in both life cycle stages. Transcription of the LmGT genes in promastigotes and axenically cultured amastigotes occurs at similar levels, as measured by nuclear run-on transcription. Consequently, the approximately 15-fold up-regulation of LmGT2 mRNA levels in promastigotes compared with amastigotes must be controlled at the post-transcriptional level. Measurement of LmGT2 RNA decay in promastigotes and axenic amastigotes treated with actinomycin D suggests that differential mRNA stability may play a role in regulating glucose transporter mRNA levels in the two life cycle stages.

Regulation of Glucose Transport, and Glucose Transporters Expression and Trafficking in the Heart: Studies in Cardiac Myocytes

Cardiac muscle is characterized by a high rate of glucose consumption. In the absence of insulin, glucose transport into cardiomyocytes limits the rate of glucose utilization and therefore it is important to understand the regulation of glucose transporters. Cardiac muscle cells express 2 distinct glucose transporters, GLUT4 and GLUT1; although GLUT4 is quantitatively the more important glucose transporter expressed in heart, GLUT1 is also expressed at a substantial level. In isolated rat cardiomyocytes, insulin acutely stimulates glucose transport and translocates both GLUT4 and GLUT1 from an intracellular site to the cell surface. Recent evidence indicates the existence of at least 2 distinct intracellular membrane populations enriched in GLUT4 with a different protein composition. Elucidation of the intracellular location of these 2 GLUT4 vesicle pools in cardiac myocytes, their role in GLUT4 trafficking, and their relation to insulin-induced GLUT4 translocation needs to be addressed.

Analysis of the glucose transporter content of islet cell lines: Implications for glucose-stimulated insulin release

Glucose transport across the plasma membrane of mammalian cells is mediated by a family of homologous proteins. Each glucose transporter isoform has a specific tissue distribution which relates to that tissue's demand for glucose. The β-cells of pancreatic islets are known to express a distinct glucose transporter isoform, termed GLUT 2, which has a high K m for glucose. In this study, we examined the glucose transporter content of normal rat islets and three beta cell lines, β-TC, HIT and RIN cells. We show that at the protein level, GLUT 2 is the only detectable transporter isoform in normal islets, and that all three cell lines also express detectable GLUT 2. In contrast, all three cell lines expressed high levels of GLUT 1, but this isoform was not detected in normal islets. Neither the native islets nor any of the cell lines expressed GLUT 3. The insulin-responsive glucose transporter GLUT 4 was detected at very low levels in β-TC cells; to our knowledge, this is the only non-muscle or adipose cell line which expresses this isoform. We propose that the elevated level of GLUT 1 expression, together with a reduced expression of the high K m transporter GLUT 2, may account for the characteristics aberrant patterns of glucose-stimulated insulin release in cell lines derived from β-cells.

The regulation of two distinct glucose transporter (GLUT1 and GLUT4) gene expressions in cultured rat thyroid cells by thyrotropin.

We investigated the glucose transporter mRNAs expressed in FRTL5, a rat thyroid cell line, and their regulation by TSH by means of the polymerase chain reaction. FRTL5 cells as well as rat thyroid tissue expressed three types of glucose transporter mRNAs: GLUT1 or erythrocyte/HepG2/brain isoform, GLUT2 or pancreatic beta-cell/liver isoform, and GLUT4 or muscle/fat isoform. GLUT1 mRNA predominated, GLUT4 mRNA was minor, and GLUT2 mRNA expression was faint. Incubation of FRTL5 cells with TSH induced a time- and concentration-dependent increase in GLUT1 mRNA levels, while GLUT4 mRNA levels were decreased. The response of GLUT1 mRNA to TSH was evident at 3 h, and the maximal response was achieved at 12 h. TSH at a dose of 1 mU/ml elicited an approximately 3-fold increase in GLUT1 mRNA levels. (Bu)2cAMP (1 mM), 8-bromo-cAMP (1 mM), and forskolin (50 microM) mimicked the effect of TSH on GLUT1 and GLUT4 mRNA levels. The increase in GLUT1 mRNA by TSH was correlated with the increase in GLUT1 protein and the increase in 2-deoxyglucose transport activity. These observations suggest that in thyroid cells, TSH stimulates glucose transport at least in part by enhancing GLUT1 gene expression, and that the effect of TSH on GLUT1 and GLUT4 mRNA levels is mediated by a cAMP-dependent pathway.

The regulation of two distinct glucose transporter (GLUT1 and GLUT4) gene expressions in cultured rat thyroid cells by thyrotropin

The regulation of two distinct glucose transporter (GLUT1 and GLUT4) gene expressions in cultured rat thyroid cells by thyrotropin

Glucose transport by cultured human fibroblasts: regulation by phorbol esters and insulin

The regulation of 3-O-methyl-d-glucose (OMG) uptake by insulin and phorbol esters was studied in culture human skin fibroblasts. Insulin rapidly stimulated OMG uptake through a mechanism independent of new protein synthesis. Maximal insulin effect was reached in 30 min and remained constant up to 12 h. The protein kinase C activators 2-O-tetradecanoyl phorbol 13-acetate (TPA) and phorbol 12,13-dibutyrate (PdBU) promoted an initial rapid stimulation followed by a secondary long-term rise of OMG influx. This latter effect of phorbol esters on OMG influx began after 1 h, reached a maximum in 12–15 h, and was prevented by the simultaneous addition of protein synthesis inhibitors, suggesting that phorbol esters increased the synthesis of new glucose transporters. In accord with this interpretation, phorbol esters, but not insulin, increased mRNA levels for two distinct glucose transporters (GLUT1 and GLUT3) in human fibroblasts. Both the rapid and the long-term effects of phorbol esters on OMG influx were dose-dependent and half-maximal stimulations occurred at 15 nM for both PdBU and TPA. Kinetic analysis of OMG uptake indicated that both effects of phorbol esters were associated with an increase in the Vmax of the transport process, with not significant changes of the Km (4–6 mM). These results suggest that, in human fibroblasts, phorbol esters, unlike insulin, produce a long-term stimulation of OMG uptake, which is dependent upon protein synthesis and is associated with increased levels of GLUT1 and GLUT3 mRNA.

Thyroid Hormone Increases Muscle/Fat Glucose Transporter Gene Expression in Rat Skeletal Muscle*

The enhancement of metabolic rate by thyroid hormone in target tissues is accompanied by increased glucose utilization, which in skeletal muscle and many other tissues is regulated at the level of membrane transport. To elucidate the role of thyroid hormone as a regulator of glucose transport in skeletal muscle, we have examined its effects on the expression of the muscle/fat (insulin-regulatable) glucose transporter, a distinct glucose transporter isotype whose expression is limited to insulin-sensitive tissues and which appears to be the major glucose transport protein in skeletal muscle. Treatment of hypothyroid rats with L-T3 (T3; 100 micrograms/100 g BW.day for 6 days) increased the abundance of the muscle/fat glucose transporter protein in crude membranes from hindlimb muscle, assessed by immunoblotting, to 145 +/- 8% of the control value in animals fasted for 17-20 h before death and to 159 +/- 16% of the control value in animals fed ad libitum (mean +/- SE). In purified plasma membranes, this effect of thyroid hormone was greater (208 +/- 23% and 219 +/- 13% of control in fasted and fed animals, respectively). Similar increases were found in an intracellular membrane fraction. Levels of muscle/fat glucose transporter mRNA were also increased in hindlimb muscle from the T3-treated rats, to 261 +/- 17% and 316 +/- 23% of control values in fasted and fed animals, respectively. In contrast, the erythroid glucose transporter protein and its mRNA, which are only weakly expressed in skeletal muscle but strongly expressed in brain, were not substantially increased by T3 treatment in either tissue. In experiments comparing hypothyroid, euthyroid, and chronically T4-treated animals, muscle/fat glucose transporter protein and mRNA abundance in skeletal muscle were also found to be dependent on thyroid status. Thyroid hormone may, thus, be an important regulator of the muscle/fat glucose transporter in skeletal muscle.

Divergent molecular mechanisms for insulin-resistant glucose transport in muscle and adipose cells in vivo.

Glucose homeostasis depends on regulated changes in glucose transport in insulin-responsive tissues (e.g. muscle and adipose cells). This transport is mediated by at least two distinct glucose transporters: "adipose-muscle" and "erythrocyte-brain." To understand the molecular basis for in vivo insulin resistance we investigated the effects of fasting and refeeding on the expression of these two glucose transporters in adipose cells and skeletal muscle. In vivo insulin resistance seen with fasting and hyperresponsiveness seen with refeeding influence glucose transporter expression in a transporter-specific and tissue-specific manner. In adipose cells only the adipose-muscle glucose transporter mRNA and protein decrease dramatically with fasting and increase above control levels with refeeding, changes that parallel effects on insulin-stimulated glucose transport. In contrast, in muscle expression of both glucose transporters increase with fasting and return to control levels with refeeding, also in accordance with changes in glucose uptake in vitro. Although expression of the adipose-muscle glucose transporter predicts the physiological response at the tissue level, factors in the hormonal/metabolic milieu appear to override its increased expression in muscle resulting in insulin-resistant glucose uptake in this tissue in vivo.

Glucose transport and microvillus membrane physical properties along the crypt-villus axis of the rabbit.

Both transport function and microvillus membrane physical properties evolve as the enterocyte matures and migrates up the crypt-villus axis. We isolated enriched fractions of villus tip, mid-villus, and crypt enterocytes from which microvillus membrane vesicles were prepared. Using this material we characterized the alterations that occur in microvillus membrane fluidity as the rabbit enterocyte matures and correlated these with kinetic studies of glucose transport. With increasing maturity the microvillus membrane becomes more rigid due to both an increase in the cholesterol/phospholipid ratio and alterations in individual phospholipid subclasses. Maximal rates of glucose transport were greatest in microvillus membrane vesicles prepared from mature cells. However, the glucose concentration producing half-maximal rates of transport (Km) was significantly lower in crypt microvillus membrane vesicles, suggesting that a distinct glucose transporter existed in crypt enterocytes. This distinction disappeared when differences between membrane lipid environments were removed. By fluidizing villus-tip microvillus membrane vesicles, in vitro, to levels seen in the crypt microvillus membrane, we observed a reduction in the Km of this transport system. These data suggest that the kinetic characteristics of the sodium-dependent glucose transporter are dependent upon its local membrane environment.

Glucose Transport and Glucose Transporters in Muscle and Their Metabolic Regulation

Skeletal muscle is the primary tissue responsible for insulin-dependent glucose uptake in vivo; therefore, glucose uptake by this tissue plays an important role in determining glycemia. Glucose uptake in muscle occurs by a system of facilitated diffusion involving at least two distinct glucose transporters, GLUT-1 and GLUT-4. Both bind the fungal metabolite and inhibitor of glucose transport cytochalasin B. In human skeletal muscle, both types of transporters are detected immunologically, and corresponding mRNA transcripts of both transporter forms are detected. In human skeletal muscle cells in culture, in which contamination by other tissues is ruled out, a 50,000-Mr polypeptide is photolabeled with cytochalasin B. In rat skeletal muscle, acute treatment with insulin in vivo increases glucose-transport activity and the number of specific cytochalasin B-binding sites at the plasma membrane. In mildly diabetic (streptozocin-induced) rats, the number of cytochalasin B-binding sites is decreased in total membranes, and preferentially in the plasma membrane. In response to acute insulin treatment, however, there is still recruitment of glucose transporters to the plasma membrane from an intracellular membrane store. Hence, migration of transporters does occur in this form of diabetes. In L6 muscle cells in culture, acute treatment (1 h) with insulin causes recruitment of glucose transporters to the plasma membrane, and prolonged exposure to insulin or to glucose-deprived medium causes increased expression of GLUT-1 mRNA and GLUT-1 protein. Prolonged exposure (24 h) to high glucose in the medium causes a decrease in the number of glucose transporters in the plasma membrane. Hence, in those cells the expression of the GLUT-1 glucose transporter is modulated by insulin.

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.

Differential regulation of two distinct glucose transporter species expressed in 3T3-L1 adipocytes: effect of chronic insulin and tolbutamide treatment.

The HepG2-type glucose transporter (HepG2-GT) is expressed in 3T3-L1 fibroblasts and adipocytes. In contrast, the acutely insulin-regulatable glucose transporter (IRGT) is expressed only in the adipocytes. In the present study, the expression of the IRGT was shown to increase in parallel with the acquisition of acutely insulin-stimulated glucose uptake during differentiation of these cells, whereas the level of the HepG2-GT decreased during the course of differentiation in parallel with a decline in basal glucose uptake. We examined the effects of chronic insulin and tolbutamide treatment on glucose transporter activity in conjunction with the expression of these two glucose transporter species in 3T3-L1 adipocytes. Treatment of adipocytes with insulin, tolbutamide, or both agents in combination increased 2-deoxyglucose uptake, HepG2-GT protein, and HepG2-GT mRNA levels in parallel. The effect of combined insulin/tolbutamide administration on these three parameters was greater than the effect of either treatment alone. In contrast, these treatments either had no significant effect or decreased levels of IRGT protein and mRNA. We conclude that chronic treatment of 3T3-L1 adipocytes with insulin or tolbutamide increases glucose uptake primarily by means of a selective increase in the expression of the HepG2-GT. We suggest that part of the in vivo hypoglycemic effect of insulin and sulfonylureas may involve an increased expression of the HepG2-GT.

Evidence for functionally distinct glucose transporters in basal and insulin-stimulated adipocytes.

The activity and Km of glucose transport of rat adipocytes are quite variable in the basal state. This could be due to differing levels of highly saturable transport against a background of less saturable transport. Such heterogeneity could lead to differing conclusions as to the Km of basal cells compared to insulin-stimulated cells depending on the choice of substrate, the range of concentrations tested, and the rigor of data analysis. In the present work, we used a cell preparation which was stable and partially activated by constant agitation. We used a two-component model to fit the concentration dependence of D-glucose uptake. We defined two parallel pathways of glucose entry, a high-affinity/low-capacity pathway and a low-affinity/high-capacity pathway. Both pathways were stereospecific and were inhibited by cytochalasin B. The low-affinity pathway in basal cells had 97% of the total capacity (Vmax) with a high Km (greater than 50 mM). A second pathway had a very low Km (less than 1 mM) and only 3% of the total capacity, but contributed to 30-60% of glucose uptake at 8 mM glucose. In insulin-stimulated cells, a pathway with a Km of 4-5 mM dominated and contributed 85% of glucose transport. The low-affinity but not the very high affinity pathway persisted in stimulated cells, but its contribution was only 10-15% of transport at 8 mM glucose. These results suggest the presence of at least two functionally distinct transporters whose respective contributions can be characterized by nonlinear regression of data over a wide range of glucose concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)

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.