Biomedical subjects

S W Cushman

Publications and source records attributed to S W Cushman.

At least 37 records · Page 2Linked to original sources

PubMed1998

Calorie restriction increases cell surface GLUT-4 in insulin-stimulated skeletal muscle.

Reduced calorie intake [calorie restriction (CR); 60% of ad libitum (AL)] leads to enhanced glucose transport without altering total GLUT-4 glucose transporter abundance in skeletal muscle. Therefore, we tested the hypothesis that CR (20 days) alters the subcellular distribution of GLUT-4. Cell surface GLUT-4 content was higher in insulin-stimulated epitrochlearis muscles from CR vs. AL rats. The magnitude of this increase was similar to the CR-induced increase in glucose transport, and GLUT-4 activity (glucose transport rate divided by cell surface GLUT-4) was unaffected by diet. The CR effect was specific to the insulin-mediated pathway, as evidenced by the observations that basal glucose transport and cell surface GLUT-4 content, as well as hypoxia-stimulated glucose transport, were unchanged by diet. CR did not alter insulin's stimulation of insulin receptor substrate (IRS)-1-associated phosphatidylinositol 3-kinase (PI3K) activity. Muscle abundance of IRS-2 and p85 subunit of PI3K were unaltered by diet, but IRS-1 content was lower in CR vs. AL. These data demonstrate that, despite IRS-1-PI3K activity similar to AL, CR specifically increases insulin's activation of glucose transport by enhancing the steady-state proportion of GLUT-4 residing on the cell surface.

3-O-Methylglucose↗

PubMed1998

Short-term exercise enhances insulin-stimulated GLUT-4 translocation and glucose transport in adipose cells.

This investigation examined the effects of short-term exercise training on insulin-stimulated GLUT-4 glucose transporter translocation and glucose transport activity in rat adipose cells. Male Wistar rats were randomly assigned to a sedentary (Sed) or swim training group (Sw, 4 days; final 3 days: 2 x 3 h/day). Adipose cell size decreased significantly but minimally (approximately 20%), whereas total GLUT-4 increased by 30% in Sw vs. Sed rats. Basal 3-O-methyl-D-[14C]glucose transport was reduced by 62%, whereas maximally insulin-stimulated (MIS) glucose transport was increased by 36% in Sw vs. Sed rats. MIS cell surface GLUT-4 photolabeling was 44% higher in the Sw vs. Sed animals, similar to the increases observed in MIS glucose transport activity and total GLUT-4. These results suggest that increases in total GLUT-4 and GLUT-4 translocation to the cell surface contribute to the increase in MIS glucose transport with short-term exercise training. In addition, the results suggest that the exercise training-induced adaptations in glucose transport occur more rapidly than previously thought and with minimal changes in adipose cell size.

Adipose Tissue↗

PubMed1998

Role of SNARE's in the GLUT4 translocation response to insulin in adipose cells and muscle.

Insulin stimulates glucose transport in skeletal muscle, heart, and adipose tissue by promoting the appearance of GLUT4, the major glucose transporter isoform present in these tissues, on the cell surface. This is achieved by differentially modulating GLUT4 exocytosis and endocytosis, between a specialized intracellular compartment and the plasma membrane. Ligands which activate the heterotrimeric GTP-binding proteins Gs and Gi appear to modulate insulin-stimulated glucose transport through effects on the fusion of docked GLUT4-containing vesicles with the plasma membrane. In insulin resistance states, reduced cellular GLUT4 levels in adipose cells fully account for the decreased glucose transport response to insulin in these cells. In contrast, although insulin-stimulated GLUT4 translocation is also impaired in muscle, total cellular levels of GLUT4 are not altered. The defect in muscle has been attributed to a GLUT4 trafficking problem and thus studies of this mechanism could provide clues as to the nature of the impairment. The movement of GLUT4-containing vesicles from an intracellular storage site to the plasma membrane and the fusion of docked GLUT4-containing vesicles with the plasma membrane are conceptually similar to some secretory processes. A general hypothesis called the SNARE hypothesis (soluble NSF attachment protein receptors where NSF stands for N-ethylmaleimide-sensitive fusion protein) postulates that the specificity of secretory vesicle targeting is generated by complexes that form between membrane proteins on the transport vesicle (v-SNARE's) and membrane proteins located on the target membrane (t-SNARE's). Several v- and t-SNARE's have been identified in adipose cells and muscle. VAMP2 and VAMP3/cellubrevin (v-SNARE's) have been shown to interact with the t-SNARE's syntaxin 4 and SNAP-23. The cytosolic protein NSF has the characteristic of binding to the v-/t-SNARE complex through its interaction with alpha-SNAP, another soluble factor. Furthermore, recent studies have demonstrated that VAMP2/3, syntaxin 4, SNAP-23, and NSF are functionally involved in insulin-stimulated GLUT4 translocation in adipose cells and thus are likely to be involved in the Gs- and Gi-mediated modulation of the glucose transport response to insulin as well. This review summarizes recent advances on the normal mechanism of GLUT4 translocation and discusses how this process could be affected in insulin resistant states such as type II diabetes.

Adipocytes↗

PubMed1997

Role of insulin receptor substrate-1 and pp60 in the regulation of insulin-induced glucose transport and GLUT4 translocation in primary adipocytes.

In muscle and fat, glucose transport occurs through the translocation of GLUT4 from an intracellular pool to the cell surface. Phosphatidylinositol (PI) 3-kinase has been shown to be required in this process. Insulin is thought to activate this enzyme by stimulating its association with tyrosine-phosphorylated proteins such as insulin receptor substrate (IRS)-1, IRS-2, Grb2-associated binder-1, and pp60. To study the role of these endogenous substrates in glucose transport, we analyzed adipocytes from IRS-1 null mice that we previously generated (Tamemoto, H., Kadowaki, T., Tobe, K., Yagi, T., Sakura, H., Hayakawa, T., Terauchi, Y., Ueki, K., Kaburagi, Y., Satoh, S., Sekihara, H., Yoshioka, S., Horikoshi, H., Furuta, Y. , Ikawa, Y., Kasuga, M., Yazaki Y., and Aizawa S. (1994) Nature 372, 182-186). In adipocytes from these mice, we showed that: 1) insulin-induced PI 3-kinase activity in the antiphosphotyrosine immunoprecipitates was 54% of wild-type; 2) pp60 was the major tyrosine-phosphorylated protein that associated with PI 3-kinase, whereas tyrosine phosphorylaion of IRS-2 as well as its association with this enzyme was almost undetectable; and 3) glucose transport and GLUT4 translocation at maximal insulin stimulation were decreased to 52 and 68% of those from wild-type. These data suggest that both IRS-1 and pp60 play a major role in insulin-induced glucose transport in adipocytes, and that pp60 is predominantly involved in regulating this process in the absence of IRS-1.

Adipocytes↗

PubMed1997

Vp165 and GLUT4 share similar vesicle pools along their trafficking pathways in rat adipose cells.

vp165 (or gp160) is an aminopeptidase that has been identified as one of the major proteins of the GLUT4-containing vesicles. In the present study we have determined the degree of co-localization between vp165 and GLUT4 in rat adipose cells and used perturbation by wortmannin to assess the exocytic and endocytic steps along the translocation and recycling pathways of GLUT4 in the absence and presence of insulin. Western blots of subcellular membrane fractions demonstrate very similar distributions of vp165 and GLUT4. Confocal microscopy of whole cells provides direct evidence that these proteins share the same vesicle populations moving both towards and from the plasma membrane. These data are consistent with the presence of a distinct insulin-sensitive compartment that sequesters both GLUT4 and vp165 and suggest similar trafficking routes through the recycling compartments.

Adipose Tissue↗

PubMed1997

Regulation of cell surface GLUT4 in skeletal muscle of transgenic mice.

Marked overexpression of the glucose transporter GLUT4 in skeletal muscle membrane fractions of GLUT4 transgenic (TG) mice is accompanied by disproportionately small increases in basal and insulin-stimulated glucose transport activity. Thus we have assessed cell surface GLUT4 by photolabelling with the membrane-impermeant reagent 2-N-[4-(1-azi-2,2,2-trifluoroethyl)benzoyl]-1, 3-bis(D-mannos-4-yloxy)-2-propylamine (ATB-BMPA) and measured the corresponding glucose transport activity using 2-deoxyglucose in isolated extensor digitorum longus (EDL) muscles from non-transgenic (NTG) and GLUT4 TG mice in the absence and presence of 13.3 nM (2000 mu units/ml) insulin, without or with hypoxia as a model of muscle contraction. TG mice displayed elevated rates of glucose transport activity under basal and insulin-stimulated conditions, and in the presence of insulin plus hypoxia, compared with NTG mice. Photoaffinity labelling of cell surface GLUT4 indicated corresponding elevations in plasma membrane GLUT4 in the basal and insulin-stimulated states, and with insulin plus hypoxia, but no difference in cell surface GLUT4 during hypoxia stimulation. Subcellular fractionation of hindlimb muscles confirmed the previously observed 3-fold overexpression of GLUT4 in the TG compared with the NTG mice. These results suggest that: (1) alterations in glucose transport activity which occur with GLUT4 overexpression in EDL muscles are directly related to cell surface GLUT4 content, regardless of the levels observed in the corresponding subcellular membrane fractions, (2) while overexpression of GLUT4 influences both basal and insulin-stimulated glucose transport activity, the response to hypoxia/ contraction-stimulated glucose transport is unchanged, and (3) subcellular fractionation provides little insight into the subcellular trafficking of GLUT4, and whatever relationship is demonstrated in EDL muscles from NTG mice is disrupted on GLUT4 overexpression.

Affinity Labels↗

PubMed1997

Rab 3D in rat adipose cells and its overexpression in genetic obesity (Zucker fatty rat).

Members of the Rab 3 subfamily of low-molecular-mass GTP-binding proteins have been functionally implicated in regulated exocytosis. The aim of the present study was to examine the subcellular distribution of a member of this family, Rab 3D, in rat adipose cells, given the hypothesis that this protein might be involved in insulin-stimulated GLUT4 exocytosis. We show that Rab 3D immunoreactivity is associated predominantly with the high-density microsomal fraction, where the signal intensity is 3- and 7-fold greater than that in plasma membranes and low-density microsomes respectively. Rab 3D does not co-localize with GLUT4 on immuno-isolated intracellular vesicles and, unlike GLUT4, it is not redistributed in response to insulin. Thus, if Rab 3D plays a role in GLUT4 trafficking, it relies on mechanisms independent of relocation. We observed that Rab 3D is overexpressed in adipose cells of obese (fa/fa) Zucker rats, in a tissue- and isoform-specific manner. The pathophysiological significance of this defect remains elusive. This could form the molecular basis for altered adipose secretory function in obesity.

Adipose Tissue↗

PubMed1997

Effects of exercise training on glucose transport and cell surface GLUT-4 in isolated rat epitrochlearis muscle.

The effects of exercise training on maximal glucose transport activity and cell surface GLUT-4 were examined in rat epitrochlearis muscle. Five days of swim training (2 x 3 h/day) produce a significant increase in citrate synthase activity (24.5 +/- 0.6 vs. 20.1 +/- 0.7 micromol x min(-1) x g(-1)), GLUT-4 content (22.9 +/- 0.8 vs. 17.4 +/- 0.4% GLUT-4 standard), and glycogen levels (54.3 +/- 9.4 vs. 28.6 +/- 9.4 micromol/g). Maximally, insulin-stimulated glucose transport activity and cell surface GLUT-4 are increased by 55 (1.50 +/- 0.11 vs. 0.97 +/- 0.10 micromol x ml(-1) x 20 min(-1)) and 48% [12.0 +/- 0.8 vs. 8.1 +/- 0.9 disintegrations x min(-1) (dpm) x mg(-1)], respectively, in exercise-trained epitrochlearis muscles. In contrast, hypoxia-stimulated glucose transport activity and cell surface GLUT-4 are reduced by 38 (0.78 +/- 0.08 vs.1.25 +/- 0.14 micromol x ml(-1) x 20 min(-1)) and 40% (5.7 +/- 0.9 vs. 9.4 +/- 1.2 dpm/mg), respectively, in exercise-trained epitrochlearis muscles. These results demonstrate that changes in insulin- and hypoxia-stimulated glucose transport activity after exercise training are fully accounted for by the appearance of cell surface GLUT-4 and support the concept of two intracellular pools of GLUT-4. Finally, we propose that high levels of muscle glycogen with exercise training may contribute to the decrease in hypoxia-stimulated glucose transport activity.

Animals↗

PubMed1997

Exercise training reverses insulin resistance in muscle by enhanced recruitment of GLUT-4 to the cell surface.

The effects of exercise training on cell surface GLUT-4 in skeletal muscle of the obese (fa/fa) Zucker rat were investigated using the impermeant glucose transporter photoaffinity reagent 2-N-4-(1-azi-2,2,2-trifluoroethyl)-benzoyl-1,3-bis- (D-mannos-4-yloxy)-2-propylamine (ATB-BMPA). In the absence of insulin, 3-O-methyl-D-glucose transport activity was no different in either fast-twitch (epitrochlearis) or slow-twitch (soleus) muscles of trained and sedentary obese rats. Likewise, basal ATB-BMPA-labeled GLUT-4 was not altered in these muscles with training. In contrast, the trained group exhibited significantly greater insulin-stimulated (2 mU/ml) glucose transport activity in epitrochlearis muscles than the sedentary group (0.53 +/- 0.03 vs. 0.18 +/- 0.03 mumol.g-1 x 10 min-1 for trained and sedentary, respectively), which was paralleled by a significant enhancement of insulin-stimulated cell surface GLUT-4 (5.33 +/- 0.20 vs. 1.57 +/- 0.14 disintegrations.min-1.mg-1 for trained and sedentary, respectively). Exercise training, however, did not alter insulin-stimulated glucose transport activity or cell surface GLUT-4 in soleus muscles. Finally, exercise training did not alter the ability of muscle contraction to elevate glucose transport activity or cell surface GLUT-4 in either epitrochlearis or soleus muscles of the obese rat. These results indicate that training improves insulin-stimulated glucose transport in muscle of the obese Zucker rat by increasing GLUT-4 content and by altering the normal intracellular distribution of these transporters such that they are now capable of migrating to the cell surface in response to the insulin stimulus.

Affinity Labels↗

PubMed1997

Immunocytochemical evidence that GLUT4 resides in a specialized translocation post-endosomal VAMP2-positive compartment in rat adipose cells in the absence of insulin.

Insulin stimulates glucose transport in rat adipose cells through the translocation of GLUT4 from a poorly defined intracellular compartment to the cell surface. We employed confocal microscopy to determine the in situ localization of GLUT4 relative to vesicle, Golgi, and endosomal proteins in these physiological insulin target cells. Three-dimensional analyses of GLUT4 immunostaining in basal cells revealed an intracellular punctate, patchy distribution both in the perinuclear region and scattered throughout the cytoplasm. VAMP2 closely associates with GLUT4 in many punctate vesicle-like structures. A small fraction of GLUT4 overlaps with TGN38-mannosidase II, gamma-adaptin, and mannose-6-phosphate receptors in the perinuclear region, presumably corresponding to late endosome and trans-Golgi network structures. GLUT4 does not co-localize with transferrin receptors, clathrin, and Igp-120. After insulin treatment, GLUT4 partially redistributes to the cell surface and decreases in the perinuclear area. However, GLUT4 remains co-localized with TGN38-mannosidase II and gamma-adaptin. Therefore, the basal compartment from which GLUT4 is translocated in response to insulin comprises specialized post-endosomal VAMP2-positive vesicles, distinct from the constitutively recycling endosomes. These results are consistent with a kinetic model in which GLUT4 is sequestered through two or more intracellular pools in series.

Adipocytes↗

PubMed1997

Insulin stimulates both leptin secretion and production by rat white adipose tissue.

Leptin, the peptide encoded by the obese gene, is secreted by adipose cells and plays a role in regulating food intake, energy expenditure, and adiposity. Because earlier studies suggested that insulin increases the expression of leptin, we investigated the effect of insulin on leptin secretion by adipose tissue. Epididymal fat pads were incubated in vitro in the presence or absence of insulin over a 4-h time course. Insulin increased leptin secretion by about 80% at all time points studied. After 10 min of insulin treatment, the amount of tissue-associated leptin was lower in insulin-stimulated tissue, presumably due to the increased secretion. At later times, both tissue-associated leptin and total leptin production were higher in insulin-treated tissue. In untreated, isolated adipose cells, immunostaining of leptin was detected in the endoplasmic reticulum by confocal microscopy. After insulin treatment, there were two populations of cells. In many cells, leptin staining became fainter and was restricted to a narrow band near the plasma membrane. However, in other cells the leptin-staining pattern was unchanged. Leptin did not colocalize with GLUT4, the glucose transporter isoform found primarily in insulin-responsive cells, in either basal or insulin-stimulated adipose cells. In this study, insulin increased both secretion and production of leptin by adipose tissue fragments. Interestingly, insulin appeared to stimulate the transport of leptin from the endoplasmic reticulum rather than acting on a pool of regulated secretory vesicles. (Endocrinology 138: 4463-4472, 1997)

Adipose Tissue↗

PubMed1997

Morphological effects of wortmannin on the endosomal system and GLUT4-containing compartments in rat adipose cells.

Studies using functional and pharmacological approaches have implicated PI 3-kinase as a key intermediate in the glucose transport and GLUT4 translocation responses to insulin. Confocal microscopy was used to investigate the effects of the PI 3-kinase inhibitor wortmannin in isolated rat adipose cells. Independent of insulin, wortmannin induces the appearance of phase-lucent vacuoles containing the endosomal markers TfR, Rab4, M6PR, and cellubrevin. When added before or with insulin, wortmannin blocks insulin-stimulated GLUT4 translocation, but does not influence the basal VAMP2-containing GLUT4 compartment. These results substantiate the concept of a specialized basal GLUT4 compartment mostly distinct from that of the recycling receptors. However, when added after insulin, wortmannin induces a rapid redistribution of GLUT4 from the cell surface into those endosomal-derived vacuoles where the GLUT4 co-localize with TfR, Rab4, cellubrevin, and VAMP2, but not with clathrin, M6PR, Golgi complex markers TGN38-mannosidase II and gamma-adaptin, and lysosomal marker lgp-120. Therefore, wortmannin also disrupts insulin-stimulated GLUT4 traffic in the recycling endosomal pathway, at a step distal to the sorting of recycling proteins from late endosomal and TGN markers; wortmannin does not appear to affect internalization from the plasma membrane, and delivery from early to late endosomes or from late endosomes to the TGN. In combination with previous kinetic biochemical studies, these results suggest that: (i) insulin stimulates the exocytosis of GLUT4 through a direct pathway from a specialized basal compartment to the plasma membrane, (ii) during endocytosis in the presence of insulin, GLUT4 is sorted out of the TfR compartment into a separate recycling pathway back to the plasma membrane, and (iii) both of these pathways involve wortmannin sensitive enzymes.

Adipose Tissue↗

PubMed1996

Identification of SNAP receptors in rat adipose cell membrane fractions and in SNARE complexes co-immunoprecipitated with epitope-tagged N-ethylmaleimide-sensitive fusion protein.

The vesicle-associated membrane proteins [VAMPs; vesicle SNAP receptors (v-SNAREs)] present on GLUT4-enriched vesicles prepared from rat adipose cells [Cain, Trimble and Lienhard (1992) J. Biol. Chem. 267, 11681-11684] have been identified as synaptobrevin 2 (VAMP 2) and cellubrevin (VAMP 3) by using isoform-specific antisera. Additional antisera identify syntaxins 2 and 4 as the predominant target membrane SNAP receptors (t-SNAREs) in the plasma membranes (PM), with syntaxin 3 at one-twentieth the level. Syntaxins 2 and 4 are enriched 5-10-fold in PM compared with low-density microsomes (LDM). Insulin treatment results in an 11-fold increase in immunodetectable GLUT4 in PM and smaller (approx. 2-fold) increases in VAMP 2 and VAMP 3, whereas the subcellular distributions of the syntaxins are not altered by insulin treatment. To determine which of the SNAP receptors (SNAREs) in PM might participate in SNARE complexes with proteins from GLUT4 vesicles, complexes were immunoprecipitated with anti-myc antibody from solubilized membranes after the addition of myc-epitope-tagged N-ethylmaleimide-sensitive fusion protein (NSF) and recombinant alpha-soluble NSF attachment protein (alpha-SNAP). These complexes contain VAMPs 2 and 3 and syntaxin 4, but not syntaxins 2 or 3. Complex formation requires ATP and is disrupted by ATP hydrolysis. When all membrane fractions are prepared from basal cells, few or no VAMPs and no syntaxin 4 are immunoprecipitated in SNARE complexes obtained from LDM alone (or from immunoisolated GLUT4 vesicles). The content of syntaxin 4 depends on the presence of PM, and participation of VAMPs 2 and 3 is enhanced 4-6-fold by the addition of solubilized GLUT4 vesicles to PM. The latter increase is greater than can be explained by the 2-fold higher levels of VAMPs added to the reaction mixture. When all membrane fractions are prepared from insulin-stimulated cells, SNARE complexes formed from PM alone contain similar levels of syntaxin 4 but 5-6-fold higher levels of VAMPs 2 and 3 compared with PM alone from basal cells. Addition of GLUT4 vesicle proteins to PM from insulin-treated cells results in a further 2-fold increase in VAMP 2 recovered in SNARE complexes. Therefore the VAMPs in PM of insulin-treated but not basal cells, and in GLUT4-vesicles from cells in either condition, are in a form that readily forms a SNARE complex with PM t-SNAREs and NSF. Insulin seems to activate PM and/or GLUT4 vesicles so as to increase the efficiency of SNARE complex formation.

Adipose Tissue↗

PubMed1996

Effects of overexpressing wild-type and mutant PDGF receptors on translocation of GLUT4 in transfected rat adipose cells.

Activation of phosphatidylinositol 3-kinase (PI3K) by insulin is necessary for the effect of insulin to recruit GLUT4 to the cell surface in insulin target cells. In adipose cells, stimulation of endogenous PDGF receptors (PDGF-R) results in increased PI3K activity without causing recruitment of GLUT4. We overexpressed wild-type or mutant forms of the PDGF-R in rat adipose cells and examined their effects on PDGF- and insulin-stimulated recruitment of co-transfected epitope-tagged GLUT4. Control cells expressing only tagged GLUT4 had a 3-fold increase in cell surface GLUT4 upon insulin stimulation but no response to PDGF. Cells overexpressing wild-type PDGF-R maintained insulin responsiveness and, in addition, acquired the ability to recruit GLUT4 in response to PDGF. Surprisingly, overexpression of F740/ F751 (mutant PDGF-R unable to directly activate PI3K) led to similar results. Nevertheless, wortmannin (an inhibitor of PI3K) blocked effects of both PDGF and insulin to recruit GLUT4. Our data suggest that overexpression of PDGF-R mediates positive effects on GLUT4 translocation by a wortmannin sensitive pathway not dependent on direct interaction of the PDGF-R with PI3K.

Adipose Tissue↗

PubMed1996

Unique subcellular distribution of five annexins in resting and insulin-stimulated rat adipose cells.

Several lines of evidence suggest that annexins, a family of phospholipid-binding proteins, play a role in cellular trafficking. Five annexins (I, II, V, VI, VII) were detected in rat adipose cells. They were primarily associated with the plasma membrane in a calcium-dependent manner. None of them redistributed with insulin treatment of the cells, in contrast to the glucose transporter GLUT4, which moved from intracellular membranes to the plasma membrane. Although the actual function of annexins in adipose cells remains to be determined, our data indicate that insulin-stimulated GLUT4 trafficking does not rely on a change in subcellular location of any of the five annexins detected so far in these cells.

Adipose Tissue↗

PubMed1996

Hormonal regulation of glucose transport in a brown adipose cell preparation isolated from rats that shows a large response to insulin.

Isolated brown adipose cells from rats are prepared whose viability is indicated by the expected stimulation of oxygen consumption by noradrenaline and counter-regulation of this oxygen consumption response by insulin. Insulin stimulates 3-O-methyl-D-glucose transport by approx. 15-fold in the absence of adenosine, and adenosine augments this response at least 2-fold. The insulin-stimulated translocation of the glucose transporter GLUT4 from an intracellular compartment to the plasma membrane is readily detected by subcellular fractionation and Western blotting, and the appearance of GLUT4 on the cell surface in response to insulin is demonstrated by bis-mannose photolabelling. Isoprenaline also stimulates glucose transport activity but only by approx. 3-fold; this effect is not altered by adenosine. Isoprenaline increases insulin-stimulated glucose transport activity in the absence of adenosine but decreases it in the presence of adenosine. These results demonstrate that although the regulation of glucose transport by insulin in brown adipose cells is qualitatively similar to that in white adipose cells, counter-regulation by adenosine and isoprenaline is at least quantitatively and may be qualitatively different. Isolated brown adipose cells from rats thus represent an excellent model for further examination of the mechanism by which multiple hormone signalling pathways interact to control glucose transport and GLUT4 subcellular trafficking.

Adenosine↗

PubMed1996

Glucose transport and GLUT4 protein distribution in skeletal muscle of GLUT4 transgenic mice.

The aim of the present investigation was to determine whether the subcellular distribution and insulin-stimulated translocation of the GLUT4 isoform of the glucose transporter are affected when GLUT4 is overexpressed in mouse skeletal muscle, and if the overexpression of GLUT4 alters maximal insulin-stimulated glucose transport and metabolism. Rates of glucose transport and metabolism were assessed by hind-limb perfusion in GLUT4 transgenic (TG) mice and non-transgenic (NTG) controls. Glucose-transport activity was determined under basal (no insulin), submaximal (0.2 m-unit/ml) and maximal (10 m-units/ml) insulin conditions using a perfusate containing 8 mM 3-O-methyl-D-glucose. Glucose metabolism was quantified by perfusing the hind limbs for 25 min with a perfusate containing 8 mM glucose and 10 m-units/ml insulin. Under basal conditions, there was no difference in muscle glucose transport between TG (1.10 +/- 0.10 mumol/h per g; mean +/- S.E.M.) and NTG (0.93 +/- 0.16 mumol/h per g) mice. However, TG mice displayed significantly greater glucose-transport activity during submaximal (4.42 +/- 0.49 compared with 2.69 +/- 0.33 mumol/h per g) and maximal (11.68 +/- 1.13 compared with 7.53 +/- 0.80 mumol/h per g) insulin stimulation. Nevertheless, overexpression of the GLUT4 protein did not alter maximal rates of glucose metabolism. Membrane purification revealed that, under basal conditions, plasma-membrane (approximately 12-fold) and intracellular-membrane (approximately 4-fold) GLUT4 protein concentrations were greater in TG than NTG mice. Submaximal insulin stimulation did not increase plasma-membrane GLUT4 protein concentration whereas maximal insulin stimulation increased this protein in both NTG (4.1-fold) and TG (2.6-fold) mice. These results suggest that the increase in insulin-stimulated glucose transport following overexpression of the GLUT4 protein is limited by factors other than the plasma-membrane GLUT4 protein concentration. Furthermore, GLUT4 overexpression is not coupled to glucose-metabolic capacity.

Animals↗

PubMed1996

Immunohistochemical localization of facilitated-diffusion glucose transporters in rat pancreatic islets.

The subcellular localization of five isoforms of facilitated-diffusion glucose transporters (GLUTs), from GLUT1 to GLUT5, in rat pancreatic islets was studied by immunohistochemistry using rabbit polyclonal antisera against mouse or rat GLUT peptides. Animals were perfusion-fixed with phosphate-buffered 4% paraformaldehyde and the pancreases were removed. Some specimens were embedded in paraffin, serially sectioned, and immunostained for glucagon, insulin, somatostatin, and the GLUTs for light microscopic observation. Others were prepared for immunoelectron microscopy by the post-embedding method. By these methods, GLUT2 immunostaining was observed on the lateral membranes of pancreatic beta-cells, whereas GLUT3 immunoreaction was predominantly localized in the cytoplasm of beta-cells and was not found in alpha-cells. In contrast, GLUT5 immunostaining was preferentially localized in the cytoplasm of alpha-cells compared to that of beta-cells. However, GLUT1 and GLUT4 were either barely or not at all detectable in any cells. These results suggest that rat islets take up glucose by at least three different processes and that blood glucose levels could be modulated differentially by: a high Km glucose transporter, GLUT2, in beta-cells; by a low Km glucose transporter, GLUT3, in beta-cells; and by a low Km glucose transporter, GLUT5, in alpha-cells.

Amino Acid Sequence↗