Biomedical subjects

S W Cushman

Publications and source records attributed to S W Cushman.

At least 19 recordsLinked to original sources

PubMed2001

Insulin-regulated trafficking of dual-labeled glucose transporter 4 in primary rat adipose cells.

In isolated rat adipose cells, physiologically relevant insulin target cells, glucose transporter 4 (GLUT4) subcellular trafficking can be assessed by transfection of exofacially HA-tagged GLUT4. To simultaneously visualize the transfected GLUT4, we fused GFP with HA-GLUT4. With the resulting chimeras, GFP-HA-GLUT4 and HA-GLUT4-GFP, we were able to visualize for the first time the cell-surface localization, total expression, and intracellular distribution of GLUT4 in a single cell. Confocal microscopy reveals that the intracellular proportions of both GFP-HA-GLUT4 and HA-GLUT4-GFP are properly targeted to the insulin-responsive aminopeptidase-positive vesicles. Dynamic studies demonstrate close similarities in the trafficking kinetics between the two constructs and with native GLUT4. However, while the basal subcellular distribution of HA-GLUT4-GFP and the response to insulin are indistinguishable from those of HA-GLUT4 and endogenous GLUT4, most of the GFP-HA-GLUT4 is targeted to the plasma membrane with little further insulin response. Thus, HA-GLUT4-GFP will be useful to study GLUT4 trafficking in vivo while GFP on the N-terminus interferes with intracellular retention.

Adipose Tissue↗

PubMed2001

Targeting of GLUT6 (formerly GLUT9) and GLUT8 in rat adipose cells.

The subcellular targeting of the two recently cloned novel mammalian glucose transporters, GLUT6 [previously referred to as GLUT9 [Doege, Bocianski, Joost and Schürmann (2000) Biochem. J. 350, 771-776] and GLUT8, was analysed by expression of haemagglutinin (HA)-epitope-tagged GLUTs in transiently transfected primary rat adipose cells. Similar to HA-GLUT4, both transporters, HA-GLUT6 and HA-GLUT8, were retained in intracellular compartments in non-stimulated cells. In contrast, mutation of the N-terminal dileucine motifs in both constructs led to constitutive expression of the proteins on the plasma membrane. Likewise, when endocytosis was blocked by co-expression of a dominant-negative mutant of the dynamin GTPase, wild-type HA-GLUT6 and HA-GLUT8 accumulated on the cell surface. However, in contrast with HA-GLUT4, no translocation of HA-GLUT6 and HA-GLUT8 to the plasma membrane was observed when the cells were stimulated with insulin, phorbol ester or hyperosmolarity. Thus GLUT6 and GLUT8 appear to recycle in a dynamin-dependent manner between internal membranes and the plasma membrane in rat adipose cells, but are unresponsive to stimuli that induce translocation of GLUT4.

Adipocytes↗

PubMed2001

The export of major histocompatibility complex class I molecules from the endoplasmic reticulum of rat brown adipose cells is acutely stimulated by insulin.

Major histocompatibility complex class I (MHC-I) molecules have been implicated in several nonimmunological functions including the regulation and intracellular trafficking of the insulin-responsive glucose transporter GLUT4. We have used confocal microscopy to compare the effects of insulin on the intracellular trafficking of MHC-I and GLUT4 in freshly isolated rat brown adipose cells. We also used a recombinant vaccinia virus (rVV) to express influenza virus hemagglutinin (HA) as a generic integral membrane glycoprotein to distinguish global versus specific enhancement of protein export from the endoplasmic reticulum (ER) in response to insulin. In the absence of insulin, MHC-I molecules largely colocalize with the ER-resident protein calnexin and remain distinct from intracellular pools of GLUT4. Surprisingly, insulin induces the rapid export of MHC-I molecules from the ER with a concomitant approximately three-fold increase in their level on the cell surface. This ER export is blocked by brefeldin A and wortmannin but is unaffected by cytochalasin D, indicating that insulin stimulates the rapid transport of MHC-I molecules from the ER to the plasma membrane via the Golgi complex in a phosphatidyl-inositol 3-kinase-dependent and actin-independent manner. We further show that the effect of insulin on MHC-I molecules is selective, because insulin does not affect the intracellular distribution or cell-surface localization of rVV-expressed HA. These results demonstrate that in rat brown adipose cells MHC-I molecule export from the ER is stimulated by insulin and provide the first evidence that the trafficking of MHC-I molecules is acutely regulated by a hormone.

Adipose Tissue, Brown↗

PubMed2001

Expression of auxilin or AP180 inhibits endocytosis by mislocalizing clathrin: evidence for formation of nascent pits containing AP1 or AP2 but not clathrin.

Although uncoating of clathrin-coated vesicles is a key event in clathrin-mediated endocytosis it is unclear what prevents uncoating of clathrin-coated pits before they pinch off to become clathrin-coated vesicles. We have shown that the J-domain proteins auxilin and GAK are required for uncoating by Hsc70 in vitro. In the present study, we expressed auxilin in cultured cells to determine if this would block endocytosis by causing premature uncoating of clathrin-coated pits. We found that expression of auxilin indeed inhibited endocytosis. However, expression of auxilin with its J-domain mutated so that it no longer interacted with Hsc70 also inhibited endocytosis as did expression of the clathrin-assembly protein, AP180, or its clathrin-binding domain. Accompanying this inhibition, we observed a marked decrease in clathrin associated with the plasma membrane and the trans-Golgi network, which provided us with an opportunity to determine whether the absence of clathrin from clathrin-coated pits affected the distribution of the clathrin assembly proteins AP1 and AP2. Surprisingly we found almost no change in the association of AP2 and AP1 with the plasma membrane and the trans-Golgi network, respectively. This was particularly obvious when auxilin or GAK was expressed with functional J-domains since, in these cases, almost all of the clathrin was sequestered in granules that also contained Hsc70 and auxilin or GAK. We conclude that expression of clathrin-binding proteins inhibits clathrin-mediated endocytosis by sequestering clathrin so that it is no longer available to bind to nascent pits but that assembly proteins bind to these pits independently of clathrin.

Adaptor Protein Complex 1↗

PubMed2001

GLUT4 overexpression in db/db mice dose-dependently ameliorates diabetes but is not a lifelong cure.

We previously reported that overexpression of GLUT4 in lean, nondiabetic C57BL/KsJ-lepr(db/+) (db/+) mice resulted in improved glucose tolerance associated with increased basal and insulin-stimulated glucose transport in isolated skeletal muscle. We used the diabetic (db/db) litter mates of these mice to examine the effects of GLUT4 overexpression on in vivo glucose utilization and on in vitro glucose transport and GLUT4 translocation in diabetic mice. We examined in vivo glucose disposal by oral glucose challenge and hyperinsulinemic-hyperglycemic clamps. We also evaluated the in vitro relationship between glucose transport activity and cell surface GLUT4 levels as assessed by photolabeling with the membrane-impermeant reagent 2-N-(4-(1-azi-2,2,2-trifluoroethyl)benzoyl)-1,3-bis(D-mannose-4-yloxy)-2-propylamine in extensor digitorum longus (EDL) muscles. All parameters were examined as functions of animal age and the level of GLUT4 overexpression. In young mice (age 10-12 weeks), both lower (two- to threefold) and higher (four- to fivefold) levels of GLUT4 overexpression were associated with improved glucose tolerance compared to age-matched nontransgenic (NTG) mice. However, glucose tolerance deteriorated with age in db/db mice, although less rapidly in transgenic mice expressing the higher level of GLUT4. Glucose infusion rates during hyperinsulinemic-hyperglycemic clamps were increased with GLUT4 overexpression, compared with NTG mice in both lower and higher levels of GLUT4 overexpression, even in the older mice. Surprisingly, isolated EDL muscles from diabetic db/db mice did not exhibit alterations in either basal or insulin-stimulated glucose transport activity or cell surface GLUT4 compared to nondiabetic db/+ mice. Furthermore, both GLUT4 overexpression levels and animal age are associated with increased basal and insulin-stimulated glucose transport activities and cell surface GLUT4. However, the observed increased glucose transport activity in older db/db mice was not accompanied by an equivalent increase in cell surface GLUT4 compared to younger animals. Thus, although in vivo glucose tolerance is improved with GLUT4 overexpression in young animals, it deteriorates with age; in contrast, insulin responsiveness as assessed by the clamp technique remains improved with GLUT4 overexpression, as does in vitro insulin action. In summary, despite an impairment in whole-body glucose tolerance, skeletal muscle of the old transgenic GLUT4 db/db mice is still insulin responsive in vitro and in vivo.

Animals↗

PubMed2000

Transient enhancement of GLUT-4 levels in rat epitrochlearis muscle after exercise training.

The purpose of the present study was to examine the effect of detraining on the glucose transport system after short-term swim training (5 days), long-term swim training (5 wk), and treadmill run training (5 wk). Skeletal muscles were isolated from female Wistar rats at 24 or 48 h posttraining. SST produces a 48% increase in GLUT-4 mRNA, a 30% increase in GLUT-4 protein, and a 60% increase in insulin-stimulated glucose transport activity at 24 h posttraining but not at 48 h posttraining. Similar to SST, long-term swim training produces a 60% increase in GLUT-4 mRNA and a 30% increase in GLUT-4 protein content at 24 h posttraining but not at 48 h posttraining. Finally, treadmill run training produces a transient 35% increase in GLUT-4 protein content that is completely reversed at 48 h after the last bout of exercise. These results demonstrate that the increase in GLUT-4 mRNA and GLUT-4 protein occurs during the first week of exercise training and is rapidly lost after training cessation. We believe that the transient enhancement in GLUT-4 protein after exercise training is due to a short GLUT-4 half-life, a process that is primarily regulated by pretranslational mechanisms.

Animals↗

PubMed2000

Demonstration of insulin-responsive trafficking of GLUT4 and vpTR in fibroblasts.

Insulin-responsive trafficking of the GLUT4 glucose transporter and the insulin-regulated aminopeptidase (IRAP) in adipose and muscle cells is well established. Insulin regulation of GLUT4 trafficking in these cells underlies the role that adipose tissue and muscle play in the maintenance of whole body glucose homeostasis. GLUT4 is expressed in a very limited number of tissues, most highly in adipose and muscle, while IRAP is expressed in many tissues. IRAP's physiological role in any of the tissues in which it is expressed, however, is unknown. The fact that IRAP, which traffics by the same insulin-regulated pathway as GLUT4, is expressed in 'non-insulin responsive' tissues raises the question of whether these other cell types also have a specialized insulin-regulated trafficking pathway. The existence of an insulin-responsive pathway in other cell types would allow regulation of IRAP activity at the plasma membrane as a potentially important physiological function of insulin. To address this question we use reporter molecules for both GLUT4 and IRAP trafficking to measure insulin-stimulated translocation in undifferentiated cells by quantitative fluorescence microscopy. One reporter (vpTR), a chimera between the intracellular domain of IRAP and the extracellular and transmembrane domains of the transferrin receptor, has been previously characterized. The other is a GLUT4 construct with an exofacial HA epitope and a C-terminal GFP. By comparing these reporters to the transferrin receptor, a marker for general endocytic trafficking, we demonstrate the existence of a specialized, insulin-regulated trafficking pathway in two undifferentiated cell types, neither of which normally express GLUT4. The magnitude of translocation in these undifferentiated cells (approximately threefold) is similar to that reported for the translocation of GLUT4 in muscle cells. Thus, undifferentiated cells have the necessary retention and translocation machinery for an insulin response that is large enough to be physiologically important.

3T3 Cells↗

PubMed2000

Immunoelectron microscopic evidence that GLUT4 translocation explains the stimulation of glucose transport in isolated rat white adipose cells.

We used an improved cryosectioning technique in combination with quantitative immunoelectron microscopy to study GLUT4 compartments in isolated rat white adipose cells. We provide clear evidence that in unstimulated cells most of the GLUT4 localizes intracellularly to tubulovesicular structures clustered near small stacks of Golgi and endosomes, or scattered throughout the cytoplasm. This localization is entirely consistent with that originally described in brown adipose tissue, strongly suggesting that the GLUT4 compartments in white and brown adipose cells are morphologically similar. Furthermore, insulin induces parallel increases (with similar magnitudes) in glucose transport activity, approximately 16-fold, and cell-surface GLUT4, approximately 12-fold. Concomitantly, insulin decreases GLUT4 equally from all intracellular locations, in agreement with the concept that the entire cellular GLUT4 pool contributes to insulin-stimulated exocytosis. In the insulin-stimulated state, GLUT4 molecules are not randomly distributed on the plasma membrane, but neither are they enriched in caveolae. Importantly, the total number of GLUT4 C-terminal epitopes detected by the immuno-gold method is not significantly different between basal and insulin-stimulated cells, thus arguing directly against a reported insulin-induced unmasking effect. These results provide strong morphological evidence (1) that GLUT4 compartments are similar in all insulin-sensitive cells and (2) for the concept that GLUT4 translocation almost fully accounts for the increase in glucose transport in response to insulin.

3-O-Methylglucose↗

PubMed1999

GLUT4 trafficking in insulin-stimulated rat adipose cells: evidence that heterotrimeric GTP-binding proteins regulate the fusion of docked GLUT4-containing vesicles.

Agents that activate the G-protein G(i) (e.g. adenosine) increase, and agents that activate G(s) [e.g. isoprenaline (isoproterenol)] decrease, steady-state insulin-stimulated glucose transport activity and cell-surface GLUT4 in isolated rat adipose cells without changing plasma membrane GLUT4 content. Here we have further examined the effects of R(s)G(s) and R(i)G(i) ligands (in which R(s) and R(i) are G(s)- and G(i)-coupled receptors respectively) on insulin-stimulated cell-surface GLUT4 and the kinetics of GLUT4 trafficking in these same cells. Rat adipose cells were preincubated for 2 min with or without isoprenaline (200 nM) and adenosine deaminase (1 unit/ml), to stimulate G(s) and decrease the stimulation of G(i) respectively, followed by 0-20 min with insulin (670 nM). Treatment with isoprenaline and adenosine deaminase decreased insulin-stimulated glucose transport activity by 58%. Treatment with isoprenaline and adenosine deaminase also resulted in similar decreases in insulin-stimulated cell-surface GLUT4 as assessed by both bis-mannose photolabelling of the substrate-binding site and biotinylation of the extracellular carbohydrate moiety when evaluated under similar experimental conditions. After stimulation with insulin in the absence of G(s) and the presence of G(i) agents, a distinct sequence of plasma membrane events took place, starting with an increase in immunodetectable GLUT4, then an increase in the accessibility of GLUT4 to bis-mannose photolabel, and finally an increase in glucose transport activity. Pretreatment with isoprenaline and adenosine deaminase before stimulation with insulin did not affect the time course of the increase in immunodetectable GLUT4 in the plasma membrane, but did delay both the increase in accessibility of GLUT4 to photolabel and the increase in glucose transport activity. These results suggest that R(s)G(s) and R(i)G(i) modulate insulin-stimulated glucose transport by influencing the extent to which GLUT4 is associated with occluded vesicles attached to the plasma membrane during exocytosis, perhaps by regulating the fusion process through which the GLUT4 in docked vesicles becomes exposed on the cell surface.

3-O-Methylglucose↗

PubMed1999

Overexpression of the glucose transporter GLUT4 in adipose cells interferes with insulin-stimulated translocation.

In adipose cells, insulin induces the translocation of GLUT4 by stimulating their exocytosis from a basal intracellular compartment to the plasma membrane. Increasing overexpression of a hemagglutinin (HA) epitope-tagged GLUT4 in rat adipose cells results in a roughly proportional increase in cell surface HA-GLUT4 levels in the basal state, accompanied by a marked reduction of the fold HA-GLUT4 translocation in response to insulin. Using biochemical methods and cotransfection experiments with differently epitope-tagged GLUT4, we show that overexpression of GLUT4 does not affect the intracellular sequestration of GLUT4 in the absence of insulin, but rather reduces the relative insulin-stimulated GLUT4 translocation to the plasma membrane. In contrast, overexpression of GLUT1 does not interfere with the targeting of GLUT4 and vice versa. These results suggest that the mechanism involved in the intracellular sequestration of GLUT4 has a high capacity whereas the mechanism for GLUT4 translocation is readily saturated by overexpression of GLUT4, implicating an active translocation machinery in the exocytosis of GLUT4.

Adipocytes↗

PubMed1999

1-[N, O-bis-(5-isoquinolinesulphonyl)-N-methyl-L-tyrosyl]-4- phenylpiperazine (KN-62), an inhibitor of calcium-dependent camodulin protein kinase II, inhibits both insulin- and hypoxia-stimulated glucose transport in skeletal muscle.

Previous studies have indicated a role for calmodulin in hypoxia-and insulin-stimulated glucose transport. However, since calmodulin interacts with multiple protein targets, it is unknown which of these targets is involved in the regulation of glucose transport. In the present study, we have used the calcium-dependent calmodulin protein kinase II (CAMKII) inhibitor 1-[N, O-bis-(5-isoquinolinesulphonyl) -N-methyl-L-tyrosyl]-4-phenylpiperazine (KN-62) to investigate the possible role of this enzyme in the regulation of glucose transport in isolated rat soleus and epitrochlearis muscles. KN-62 did not affect basal 2-deoxyglucose transport, but it did inhibit both insulin- and hypoxia-stimulated glucose transport activity by 46 and 40% respectively. 1-[N,O-Bis-(1, 5-isoquinolinesulphonyl)-N-methyl-l-tyrosyl]-4-phenylpiperazine (KN-04), a structural analogue of KN-62 that does not inhibit CAMKII, had no effect on hypoxia-or insulin-stimulated glucose transport. Accordingly, KN-62 decreased the stimulated cell-surface GLUT4 labelling by a similar extent as the inhibition of glucose transport (insulin, 49% and hypoxia, 54%). Additional experiments showed that KN-62 also inhibited insulin- and hypoxia-stimulated transport by 37 and 40% respectively in isolated rat epitrochlearis (a fast-twitch muscle), indicating that the effect of KN-62 was not limited to the slow-twitch fibres of the soleus. The inhibitory effect of KN-62 on hypoxia-stimulated glucose transport appears to be specific to CAMKII, since KN-62 did not inhibit hypoxia-stimulated 45Ca efflux from muscles pre-loaded with 45Ca, or hypoxia-stimulated glycogen breakdown. Additionally, KN-62 affected neither insulin-stimulated phosphoinositide 3-kinase nor Akt activity, suggesting that the effects of KN-62 are not due to non-specific effects of this inhibitor on these regions of the insulin-signalling cascade. The results of the present study suggest that CAMKII might have a distinct role in insulin- and hypoxia-stimulated glucose transport, possibly in the vesicular trafficking of GLUT4.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

PubMed1999

SNAP-23 participates in SNARE complex assembly in rat adipose cells.

SNARE proteins are required for vesicle docking and fusion in eukaryotic cells in processes as diverse as homotypic membrane fusion and synaptic vesicle exocytosis [SNARE stands for SNAP receptor, where SNAP is soluble NSF attachment protein]. The SNARE proteins syntaxin 4 and vesicle-associated membrane protein (VAMP) 2/3 also participate in the insulin-stimulated translocation of GLUT4 from intracellular vesicles to the plasma membrane in adipose cells. We now report the molecular cloning and characterization of rat SNAP-23, a ubiquitously expressed homologue of the essential neuronal SNARE protein SNAP-25 (synaptosomal-associated protein of 25 kDa). Rat SNAP-23 is 86% and 98% identical respectively to human and mouse SNAP-23. Southern blot analysis reveals that the rat, mouse and human SNAP-23 genes encode species-specific isoforms of the same protein. Co-immunoprecipitation of syntaxin 4 and SNAP-23 shows association of these two proteins in rat adipose cell plasma membranes, and insulin stimulation does not alter the SNAP-23/syntaxin 4 complex. In addition, we demonstrate for the first time the participation of SNAP-23, along with syntaxin 4 and VAMP2/3, in the formation of 20S SNARE complexes prepared using rat adipose cell membranes and recombinant alpha-SNAP and NSF proteins. The stoichiometry of the SNARE complexes formed is essentially identical using membranes from either unstimulated or insulin-stimulated adipose cells. These data demonstrate that rat SNAP-23 associates with syntaxin 4 before insulin stimulation and is present in the SNARE complexes known to mediate the translocation of GLUT4 from intracellular vesicles to the plasma membrane of rat adipose cells.

Adipocytes↗

PubMed1998

Analysis of GLUT4 distribution in whole skeletal muscle fibers: identification of distinct storage compartments that are recruited by insulin and muscle contractions.

The effects of insulin stimulation and muscle contractions on the subcellular distribution of GLUT4 in skeletal muscle have been studied on a preparation of single whole fibers from the rat soleus. The fibers were labeled for GLUT4 by a preembedding technique and observed as whole mounts by immunofluorescence microscopy, or after sectioning, by immunogold electron microscopy. The advantage of this preparation for cells of the size of muscle fibers is that it provides global views of the staining from one end of a fiber to the other and from one side to the other through the core of the fiber. In addition, the labeling efficiency is much higher than can be obtained with ultracryosections. In nonstimulated fibers, GLUT4 is excluded from the plasma membrane and T tubules. It is distributed throughout the muscle fibers with approximately 23% associated with large structures including multivesicular endosomes located in the TGN region, and 77% with small tubulovesicular structures. The two stimuli cause translocation of GLUT4 to both plasma membrane and T tubules. Quantitation of the immunogold electron microscopy shows that the effects of insulin and contraction are additive and that each stimulus recruits GLUT4 from both large and small depots. Immunofluorescence double labeling for GLUT4 and transferrin receptor (TfR) shows that the small depots can be further subdivided into TfR-positive and TfR-negative elements. Interestingly, we observe that colocalization of TfR and GLUT4 is increased by insulin and decreased by contractions. These results, supported by subcellular fractionation experiments, suggest that TfR-positive depots are only recruited by contractions. We do not find evidence for stimulation-induced unmasking of resident surface membrane GLUT4 transporters or for dilation of the T tubule system (Wang, W., P.A. Hansen, B.A. Marshall, J.O. Holloszy, and M. Mueckler. 1996. J. Cell Biol. 135:415-430).

Animals↗

PubMed1998

Endocytosis of the glucose transporter GLUT4 is mediated by the GTPase dynamin.

To study the role of the GTPase dynamin in GLUT4 intracellular recycling, we have overexpressed dynamin-1 wild type and a GTPase-negative mutant (K44A) in primary rat adipose cells. Transfection was accomplished by electroporation using an hemagglutinin (HA)-tagged GLUT4 as a reporter protein. In cells expressing HA-GLUT4 alone, insulin results in an approximately 7-fold increase in cell surface anti-HA antibody binding. Studies with wortmannin indicate that the kinetics of HA-GLUT4-trafficking parallel those of the native GLUT4 and in addition, that newly synthesized HA-GLUT4 goes to the plasma membrane before being sorted into the insulin-responsive compartments. Short term (4 h) coexpression of dynamin-K44A and HA-GLUT4 increases the amount of cell surface HA-GLUT4 in both the basal and insulin-stimulated states. Under conditions of maximal expression of dynamin-K44A (24 h), most or all of the intracellular HA-GLUT4 appears to be present on the cell surface in the basal state, and insulin has no further effect. Measurements of the kinetics of HA-GLUT4 endocytosis show that dynamin-K44A blocks internalization of the glucose transporters. In contrast, expression of dynamin wild type decreases the amount of cell surface HA-GLUT4 in both the basal and insulin-stimulated states. These data demonstrate that the endocytosis of GLUT4 is largely mediated by processes which require dynamin.

Adipocytes↗

PubMed1998

Effects of noradrenaline on the cell-surface glucose transporters in cultured brown adipocytes: novel mechanism for selective activation of GLUT1 glucose transporters.

Glucose transport into rat brown adipocytes has been shown to be stimulated directly by the sympathetic neurotransmitter, noradrenaline, without a significant increase in the protein content of either GLUT1 or GLUT4 glucose transporter in the plasma membrane [Shimizu, Kielar, Minokoshi and Shimazu (1996) Biochem. J. 314, 485-490]. In the present study, we labelled the exofacial glucose-binding sites of GLUT1 and GLUT4 with a membrane-impermeant photoaffinity reagent, 2-N-[4-(1-azitrifluoroethyl)benzoyl]-[2-3H]1,3-bis- (D-mannos-4-yloxy)-2-propylamine (ATB-[3H]BMPA), to determine which isoform is responsible for the noradrenaline-induced increase in glucose transport into intact brown adipocytes in culture. Insulin stimulated the rate of hexose transport by increasing ATB-[3H]BMPA-labelled cell-surface GLUT4. In contrast, the noradrenaline-induced increase in glucose transport was not accompanied by an increased ATB-[3H]BMPA labelling of GLUT4, nor with an increased amount of GLUT4 in the plasma membrane fraction as assessed by Western blotting, indicating that noradrenaline does not promote the translocation of GLUT4. However, noradrenaline induced an increase in photoaffinity labelling of cell-surface GLUT1 without an apparent increase in the immunoreactive GLUT1 protein in the plasma membrane. This is suggestive of an increased affinity of GLUT1 for the ligand. In fact, the Ki value of non-radioactive ATB-BMPA for 2-deoxy-D-glucose uptake was significantly decreased after treatment of the cells with noradrenaline. The increased photoaffinity labelling of GLUT1 and increased glucose transport caused by noradrenaline were inhibited by a cAMP antagonist, cAMP-S Rp-isomer. These results demonstrate that noradrenaline stimulates glucose transport in brown adipocytes by enhancing the functional activity of GLUT1 through a cAMP-dependent mechanism.

Adipose Tissue, Brown↗

PubMed1998

Molecular mechanisms involved in GLUT4 translocation in muscle during insulin and contraction stimulation.

Studies in mammalian cells have established the existence of numerous intracellular signaling cascades that are critical intermediates in the regulation of various biological functions. Over the past few years considerable research has shown that many of these signaling proteins are expressed in skeletal muscle. However, the detailed mechanisms involved in the regulation of glucose transporter (GLUT4) translocation from intracellular compartments to the cell surface membrane in response to insulin and contractions in skeletal muscle are not well understood. In the present essay we report three different approaches to unravel the GLUT4 translocation mechanism: 1. specific pertubation of the insulin and/or contraction signaling pathways; 2. characterization of the protein composition of GLUT4-containing vesicles with the expectation that knowledge of the constituent proteins of the vesicles may help in understanding their trafficking; 3. degree of co-immunolocalization of the GLUT4 glucose transporters with other membrane marker proteins assessed by immunofluorescense and electron microscopy.

Animals↗

PubMed1998

Dexamethasone inhibits insulin-stimulated recruitment of GLUT4 to the cell surface in rat skeletal muscle.

To test the hypothesis that glucocorticoids reduce insulin-stimulated skeletal muscle glucose transport by inhibiting the recruitment of GLUT4 glucose transporters to the cell surface, we determined the effect of glucocorticoid treatment on cell-surface GLUT4 using the impermeant glucose transporter photolabel, 2-N-4-(1-azi-2,2,2-trifluoroethyl)benzoyl-[2-3H]1,3-bis-(D-mann os-4-yloxy)-2-propylamine (ATB-[2-3H]BMPA), and GLUT4 immunoprecipitation. Male Sprague-Dawley rats were treated with dexamethasone ([Dex] 0.9 mg/kg for 2 days) and compared against pair-fed controls. 2-[3H]deoxyglucose (2-[3H]DG) uptake in isolated soleus muscles was measured under conditions in which uptake reflects glucose transport activity. In control muscles, 2-[3H]DG uptake was stimulated eightfold by insulin (20 nmol/L). Dex treatment reduced maximal insulin-stimulated 2-[3H]DG uptake by 48% +/- 4% (mean +/- SEM) and decreased cell-surface (ATB-[2-3H]BMPA-photolabeled) GLUT4 by 48% +/- 3%, despite an increase in total muscle GLUT4 content of 26% +/- 7%. These findings indicate that glucocorticoid-induced inhibition of insulin-stimulated glucose transport in muscle is due to impaired recruitment of GLUT4 to the cell surface.

Animals↗

PubMed1998

Mechanism of hypoxia-stimulated glucose transport in rat skeletal muscle: potential role of glycogen.

We have previously reported that exercise training is associated with enhanced insulin-stimulated glucose transport activity and inhibited hypoxia-stimulated glucose transport activity in rat epitrochlearis muscle. Here we examine the potential role of muscle glycogen in the inhibited glucose transport response to hypoxia. Three days of swim training (2 x 3 h/day) produce a 100% increase in glycogen and a 70% increase in GLUT-4 in epitrochlearis muscle. Glucose transport after 1 h of hypoxia in muscles from fed exercise-trained (ET) rats is not significantly elevated above basal and is 40% lower than that in muscles from fed sedentary (SED) rats. Glycogen levels after 1 h of hypoxia are reduced by 27 and 64% in muscles from fed ET and fed SED rats, respectively. After 2 h of hypoxia, glucose transport is significantly increased above basal in muscles from fed ET rats, but this response is still 55% lower than that in muscles from fed SED rats. After 2 h of hypoxia, glycogen is reduced by 50 and 83% in muscles from fed ET and fed SED rats, respectively. After a modified overnight fast (approximately 4.5 g of chow), the glucose transport and glycogen responses to 1 h of hypoxia are not significantly different between muscles from ET and SED rats. These findings demonstrate a strong inverse relationship between glycogen and hypoxia-stimulated glucose transport activity and that high levels of glycogen contribute to the inhibited glucose transport response to hypoxia. Furthermore, failure of the overexpression of GLUT-4 after exercise training to enhance the glucose transport response to contraction/hypoxia suggests selective targeting of the additional GLUT-4 to the insulin-responsive pool.

Animals↗