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Biomedical subjects

I A Simpson

Publications and source records attributed to I A Simpson.

At least 109 records · Page 6Linked to original sources
PubMed1989

Spatial velocity distribution and acceleration in serial subvalve tunnel and valvular obstructions: an in vitro study using Doppler color flow mapping.

To evaluate the spatial distribution of flow velocities, turbulence and spatial acceleration in serial tunnel-valve obstruction, Doppler color flow mapping was performed in a pulsatile flow model with a tunnel obstruction (1.0 or 1.5 cm2) inserted at 2, 20 and 40 mm proximal to a mildly stenotic bioprosthetic valve studied at flow rates of 1, 2.7 and 4.9 liters/min. Measured pressure gradients were consistently higher across the tunnel (mean +/- SD 32.7 +/- 26.5 mm Hg) than across the tunnel plus valve (28.8 +/- 26.9 mm Hg, p less than 0.01). Doppler color flow map images were analyzed using a Sony RGB video-digitizing computer, providing numerical velocity assignments for the blue, red and green (variance) pixel components to allow the flow maps to be constructed into digital velocity maps and pseudo three-dimensional velocity maps. The maximal velocity stream extended distal to the tunnel (2 to 19 mm), and the length of this extension correlated well with the pressure gradient measured across the tunnel (r = 0.89), with a rapidly decelerating and turbulent spray area seen immediately distal to the valve. Pressure gradient calculated from the maximal velocity derived from the color flow map, which could only be estimated from the velocity maps for the 1.5 cm2 tunnel, correlated well with the gradient measured across the tunnel (18.0 +/- 14.1 versus 19.2 +/- 14.5 mm Hg, respectively, r = 0.98). Acceleration was seen proximal to both tunnels.(ABSTRACT TRUNCATED AT 250 WORDS)

Blood Flow Velocity↗
PubMed1989

Regulation of rat brain/HepG2 glucose transporter gene expression by insulin and insulin-like growth factor-I in primary cultures of neuronal and glial cells.

We have demonstrated the expression of the rat brain/HepG2 glucose transporter gene in primary cultures of rat neuronal and glial cells by Northern blot analysis with a rat brain glucose transporter cDNA probe. Incubation of both neuronal and glial cells with insulin and insulin-like growth factor-I induced a time- and dose-dependent increase in the steady state levels of glucose transporter mRNA. The maximal response was achieved between 2-4 h and subsequently decreased. Both insulin and insulin-like growth factor-I at a dose of 1 ng/ml elicited an approximately 57% increase in glucose transporter mRNA levels in neuronal cultures after 90 min, suggesting that each peptide was acting through its own receptor. On the other hand, insulin stimulated [3H]2-deoxyglucose uptake in glial, but not neuronal, cells. These results suggest that insulin-like peptides regulate the expression of the rat brain/Hep G2 glucose transporter gene at both transcriptional and posttranscriptional levels, and that these regulatory mechanisms may be dissociated from each other. Insulin-like peptides may, therefore, participate in the control of brain energy metabolism.

Animals↗
PubMed1988

Insulin-induced subcellular redistribution of insulin-like growth factor II receptors in the rat adipose cell. Counterregulatory effects of isoproterenol, adenosine, and cAMP analogues.

Insulin shifts the steady-state subcellular distribution of insulin-like growth factor II (IGF-II) receptors from a large intracellular pool to the plasma membrane in the rat adipose cell (Wardzala, L. J., Simpson, I. A., Rechler, M. M., and Cushman, S. W. (1984) J. Biol. Chem. 259, 8378-8383). In the present study, the counterregulatory effects of adrenergic stimulation, adenosine deaminase, and cAMP on this process were studied. Both isoproterenol (10(-6) M) and adenosine deaminase reduced insulin sensitivity and also rapidly (t1/2 approximately 1.5 min) decreased the effect of a maximal insulin concentration on the number of cell surface IGF-II receptors by 35-50%, and by 70% when added together. The marked reduction in binding was retained in isolated and solubilized plasma membranes. Both isoproterenol and adenosine deaminase alone increased the EC50 for insulin from 0.06 to 0.17 nM and, when combined, to 0.6 nM. N6-Monobutyryl-cAMP and 8-bromo-cAMP were equally potent in reducing IGF-II binding in the absence of insulin and inhibited maximal insulin-stimulated IGF-II binding by 60 and 30%, respectively. However, only the nonhydrolyzable cAMP analogue, N6-monobutyryl-cAMP, reduced the insulin sensitivity (EC50 0.7 nM). An important stimulatory role for Gi (guanine nucleotide-binding regulatory protein that inhibits adenylate cyclase) was indicated by the altered activities of cells from pertussis toxin-treated animals. The results suggest that beta-adrenergic stimulation through a cAMP-dependent mechanism markedly alters the insulin-stimulated redistribution of IGF-II receptors. This effect is additional to the potent antagonistic action of cAMP on insulin's signalling mechanism.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1988

Characterization of the stimulatory action of insulin on insulin-like growth factor II binding to rat adipose cells. Differences in the mechanism of insulin action on insulin-like growth factor II receptors and glucose transporters.

Insulin is known to increase the number of cell surface insulin-like growth factor II (IGF-II) receptors in isolated rat adipose cells through a subcellular redistribution mechanism similar to that for the glucose transporter. The effects of insulin on these two processes, therefore, have now been directly compared in the same cell preparations. 1) Insulin increases the steady state number of cell surface IGF-II receptors by 7-13-fold without affecting receptor affinity; however, insulin stimulates glucose transport activity by 25-40-fold. 2) The insulin concentration required for half-maximal stimulation of cell surface IGF-II receptor number is approximately 30% lower than that for the stimulation of glucose transport activity. 3) The half-time for the achievement of insulin's maximal effect at 37 degrees C is much shorter for IGF-II receptor number (approximately 0.8 min) than for glucose transport activity (approximately 2.6 min). 4) Reversal of insulin's action at 37 degrees C occurs more rapidly for cell surface IGF-II receptors (t1/2 congruent to 2.9 min) than for glucose transport activity (t1/2 congruent to 4.9 min). 5) When the relative subcellular distribution of IGF-II receptors is examined in basal cells, less than 10% of the receptors are localized to the plasma membrane fraction indicating that most of the receptors, like glucose transporters, are localized to an intracellular compartment. However, in response to insulin, the number of plasma membrane IGF-II receptors increases only approximately 1.4-fold while the number of glucose transporters increases approximately 4.5-fold. Thus, while the stimulatory actions of insulin on cell surface IGF-II receptors and glucose transport activity are qualitatively similar, marked quantitative differences suggest that the subcellular cycling of these two integral membrane proteins occurs by distinct processes.

3-O-Methylglucose↗
PubMed1988

Cine magnetic resonance imaging for evaluation of congenital heart disease: role in pediatric cardiology compared with echocardiography and angiography.

Cine magnetic resonance imaging was used for preoperative and postoperative evaluation of 91 patients with congenital heart disease. Their ages ranged from 6 weeks to 14 years. The quality of study was excellent in 81% of the cases, in which it provided complete documentation of diagnostic features; substantial diagnostic information was provided in another 14%, and the study was nondiagnostic in only 5%. This technique was most useful for evaluating areas of the right ventricle (95%), great arteries (95%), vena cava (94%), and pulmonary venous system (91%). The anatomic delineation of these areas was comparable to that obtained by cineangiography and was superior to echocardiography, especially in postoperative patients. For complex venous anomalies, magnetic resonance imaging provided better anatomic details than did angiography or echocardiography. The capability of multiplanar imaging allows complex angled views through the desired planes of the heart and great vessels. Proper sedation is essential to obtain maximum diagnostic information. Our preliminary experience suggests that this new modality provides excellent anatomic information in infants and children with congenital cardiac defects and will play an increasing role in pediatric cardiac diagnosis.

Adolescent↗
PubMed1988

Assessment of coronary artery bypass graft flow by intraoperative Doppler ultrasound technique.

A high frequency (10 MHz) Doppler ultrasound pencil probe was used to estimate flow in coronary artery bypass grafts in vitro and during cardiac surgery. In vitro, increasing the angle of the Doppler transducer in relation to flow caused underestimation of the Doppler derived flow, and using the external rather than internal vessel diameter to calculate cross sectional area caused significant overestimation. This overestimation was considerably greater for the internal mammary artery. Accurate flow estimation was obtained in vitro by the Doppler technique, but considerable variation occurred in patients studied during coronary artery bypass surgery. In conclusion, intraoperative Doppler ultrasound provides a qualitative assessment of phasic flow in coronary artery bypass grafts but is less accurate in quantifying absolute flow in these patients.

Adult↗
PubMed1988

Cine magnetic resonance imaging after surgical repair in patients with transposition of the great arteries.

Cine magnetic resonance imaging (MRI) was used for postoperative evaluation of eight patients who underwent intra-atrial baffle procedure for surgical repair of D-transposition of the great arteries (D-TGA). Their ages ranged from 9 months to 8 years. Younger patients were sedated with chloral hydrate (80 to 100 mg/kg) orally. MRI was performed with use of a General Electric Signa system operating at a field strength of 1.5 tesla. A body or head coil was used depending on the size of the patient. Images were obtained by use of a technique of gradient-recalled acquisition in steady state (GRASS) that utilizes a low flip angle and shorter repetition and echo times. Five patients had widely patent venae cavae and three had superior vena caval obstruction at the junction of the right atrium with a dilated azygos vein. There was no evidence of pulmonary venous obstruction in any of the patients. Right ventricular function was assessed in four patients and their ejection fractions ranged from 58% to 81%. Tricuspid and mitral regurgitation were observed in three and two patients, respectively. Both right and left ventricular outflow tracts were well visualized and showed no evidence of obstruction. Cine MRI is an entirely noninvasive, nonionizing, and safe procedure in young patients and appears to be a valuable alternative method for evaluating patients after surgical repair of D-TGA. With advancing technologies and an accumulation of experience with cine MRI, it appears that this new technique will play an important role in patient care for children with congenital heart disease.

Child↗
PubMed1988

Color Doppler flow mapping in patients with coarctation of the aorta: new observations and improved evaluation with color flow diameter and proximal acceleration as predictors of severity.

We performed color Doppler flow mapping in 15 patients, 1 week to 17 years old (mean 42 months), with coarctation of the aorta that was confirmed subsequently by angiography and/or surgery. Twelve patients had native coarctation and three had mild recoarctation after surgical repair. Color Doppler flow maps were analyzed with a digital analysis package and a Sony computer system. The diameter in the region of coarctation from the color Doppler flow map (mean = 2.0 +/- 0.8 mm [SD]) correlated well with the coarctation diameter measured at angiography (mean = 1.8 +/- 0.8 mm; r = .83, SEE 0.43 mm) in the 10 patients with native coarctation undergoing angiography, but the coarctation diameter measured by two-dimensional echocardiography (3.9 +/- 1.5 mm) was poorly predictive of the angiographic severity (r = .23). Additionally, spatial acceleration was seen in all patients proximal to the coarctation site, with an aliased and accelerating stream narrowing progressively as it proceeded toward the coarctation site, a pattern that is not seen in healthy subjects. Computer analysis of the color Doppler images provided pseudo three-dimensional and digital velocity maps for blue, red, and green (turbulent) flow velocities to allow an enhanced appreciation of the accelerating stream, easily separating this from normal descending aortic aliasing patterns. The narrowing of the acceleration area in the proximal descending aorta (distal/proximal acceleration zone ratio) was also predictive of the angiographic severity of coarctation (r = .83). The distribution of low-level turbulence seen proximally paralleled the distribution of the proximal accelerating stream.(ABSTRACT TRUNCATED AT 250 WORDS)

Aorta↗
PubMed1988

Cine magnetic resonance imaging for evaluation of anatomy and flow relations in infants and children with coarctation of the aorta.

Sixteen cine magnetic resonance imaging (MRI) studies were performed in 14 patients aged 1 week to 17 years (mean age, 46 months), who had coarctation of the aorta confirmed at angiography or surgery. Conventional echocardiographic-gated MRI was first performed in axial and rotated sagittal views and was used to identify the slice locations for cine MRI. Cine MRI was performed by gradient-recalled acquisition in steady state with a 30 degree flip angle, 12-msec echo time, 22-msec pulse repetition time, and a 128 x 256 acquisition matrix. Coarctation anatomy was extremely well defined in all but one patient who had vascular clips at the coarctation repair site. The smallest descending aortic flow diameter on cine MRI showed excellent agreement with angiography (r = 0.90). Lucent jets of high-velocity flow through the site of coarctation were imaged in eight patients, and jet length correlated well with the angiographic severity of coarctation (r = -0.81). Two patients were restudied after surgery, and they exhibited excellent repair and normal flow patterns. Cine MRI provides high-resolution imaging of coarctation anatomy with a dynamic spatial and temporal visualization of flow and with excellent detail of vascular anatomy and flow both proximal and distal to the coarctation.

Adolescent↗
PubMed1988

Divergent mechanisms for the insulin resistant and hyperresponsive glucose transport in adipose cells from fasted and refed rats. Alterations in both glucose transporter number and intrinsic activity.

The effects of fasting and refeeding on the glucose transport response to insulin in isolated rat adipose cells have been examined using 3-O-methylglucose transport in intact cells and cytochalasin B binding and Western blotting in subcellular membrane fractions. After a 72-h fast, basal glucose transport activity decreases slightly and insulin-stimulated activity decreases greater than 85%. Following 48 h of fasting, insulin-stimulated glucose transport activity is diminished from 3.9 +/- 0.5 to 1.3 +/- 0.3 fmol/cell per min (mean +/- SEM). Similarly, the concentrations of glucose transporters are reduced with fasting in both the plasma membranes from insulin-stimulated cells from 38 +/- 5 to 18 +/- 3 pmol/mg of membrane protein and the low density microsomes from basal cells from 68 +/- 8 to 34 +/- 9 pmol/mg of membrane protein. Ad lib. refeeding for 6 d after a 48-h fast results in up to twofold greater maximally insulin-stimulated glucose transport activity compared with the control level (7.1 +/- 0.4 vs. 4.5 +/- 0.2 fmol/cell per min), before returning to baseline at 10 d. However, the corresponding concentration of glucose transporters in the plasma membranes is restored only to the control level (45 +/- 5 vs. 50 +/- 5 pmol/mg of membrane protein). Although the concentration of glucose transporters in the low density microsomes of basal cells remains decreased, the total number is restored to the control level due to an increase in low density microsomal protein. Thus, the insulin-resistant glucose transport in adipose cells from fasted rats can be explained by a decreased translocation of glucose transporters to the plasma membrane due to a depleted intracellular pool. In contrast, the insulin hyperresponsive glucose transport observed with refeeding appears to result from (a) a restored translocation of glucose transporters to the plasma membrane from a repleted intracellular pool and (b) enhanced plasma membrane glucose transporter intrinsic activity.

3-O-Methylglucose↗
PubMed1988

Activation of adenylate cyclase and inhibition of glucose transport in rat adipocytes by forskolin analogues: structural determinants for distinct sites of action.

Forskolin and four analogues of forskolin, 7-beta-[gamma-(N'-methylpiperazino)-butyryloxy]-7-desacet ylforskolin, 7-desacetylforskolin, 7-tosyl-7-desacetylforskolin, and 1,9-dideoxyforskolin, were tested for their ability to activate adenylate cyclase, inhibit glucose transport, and inhibit cytochalasin B binding in rat adipocyte membranes. Forskolin was the most potent analogue in activating adenylate cyclase with an EC50 of 2 microM, whereas 7-beta-[gamma-(N'-methylpiperazino)butyryloxy]-7-desacety lforskolin and 7-desacetylforskolin were less potent, with EC50 values of 3 microM and 20 microM, respectively. The 7-tosyl-7-desacetylforskolin and 1,9-dideoxyforskolin did not stimulate adenylate cyclase even at the highest concentrations tested (100 microM). In contrast, forskolin and all of the analogues were able to fully inhibit glucose transport in adipocyte plasma membranes. The order of potency for the inhibition was forskolin greater than 7-beta-[gamma-(N'-methylpiperazino)butyryloxy]-7-desacety lforskolin greater than 7-desacetylforskolin greater than 7-tosyl-7-desacetylforskolin greater than 1,9-dideoxyforskolin, and the EC50 values were 0.24 microM, 1.8 microM, 7.1 microM, 8.8 microM, and 12.8 microM, respectively. Cytochalasin B binding to rat adipocyte membranes was inhibited by forskolin and the four analogues with the same order of potency as observed for the inhibition of glucose transport. Thus, the site of action of forskolin which is responsible for the inhibition of glucose transport and cytochasin B binding exhibits structural requirements for forskolin and its analogues that are different from those of the site responsible for the activation of adenylate cyclase.

Adenylyl Cyclases↗
PubMed1987

Activity and phosphorylation state of glucose transporters in plasma membranes from insulin-, isoproterenol-, and phorbol ester-treated rat adipose cells.

The counterregulatory action of catecholamines on insulin-stimulated glucose transport and its relation to glucose transporter phosphorylation were studied in isolated rat adipose cells. Plasma membranes exhibiting reduced glucose transport activity were prepared as described previously (Joost, H. G., Weber, T. M., Cushman, S. W., and Simpson, I. A. (1986) J. Biol. Chem. 261, 10033-10036) from cells treated with insulin, and subsequently with isoproterenol and adenosine deaminase. In these membranes, transporter affinity for cytochalasin B binding was significantly reduced (KD = 133.5 +/- 14 versus 89.8 +/- 11 nM, means +/- S.E.) with no change in number of sites or immunoreactivity of the transporter on Western blots. Reconstituted plasma membrane transport was significantly lower with isoproterenol treatment (0.50 +/- 0.12 versus 0.97 +/- 0.27 nmol/mg protein/10 s). In contrast, transport activity reconstituted from corresponding intracellular transporters (from low density microsomes) was unchanged (5.4 +/- 2.2 versus 6.9 +/- 1.2 nmol/mg protein/10 s). Thus, the intrinsic activity change of the transporter produced by catecholamines appears to reflect a structural modification that is confined to the plasma membrane and not recycled into the intracellular compartment. In cells equilibrated with [32P]phosphate, neither insulin nor isoproterenol induced [32P]phosphate incorporation into the glucose transporter immunoprecipitated from plasma membranes. Conversely, phorbol 12-myristate 13-acetate stimulated significant incorporation of [32P]phosphate into the glucose transporter in insulin-stimulated cells without any change in plasma membrane transport activity or transporter concentration. Thus, the phosphorylation state of the glucose transporter does not seem to be involved in either signaling transporter translocation or triggering changes in transporter intrinsic activity.

Adenosine Deaminase↗
PubMed1987

Regulation of insulin-stimulated glucose transport in the isolated rat adipocyte. cAMP-independent effects of lipolytic and antilipolytic agents.

This paper examines the modulation of insulin-stimulated glucose transport activity in rat adipose cells by ligands for receptors (R) that mediate stimulation (Rs; lipolytic) or inhibition (Ri; antilipolytic) of adenylate cyclase. The changes in glucose transport activity and cAMP, as assessed by 3-O-methylglucose uptake and (-/+) cAMP-dependent protein kinase (A-kinase) activity ratios, respectively, were monitored under conditions that maintain steady-state A-kinase activity ratios (Honnor, R. C., Dhillon, G. S., and Londos, C. (1985) J. Biol. Chem. 260, 15122-15129). Removal of endogenous adenosine with adenosine deaminase decreased insulin-stimulated glucose transport activity by approximately 30%, which was prevented or restored with Ri agonists such as phenylisopropyladenosine, nicotinic acid, and prostaglandin E1. These changes in transport activity were not accompanied by changes in A-kinase activity ratios, indicating that Ri-mediated effects on transport are independent of cAMP changes. Addition of an Rs ligand, isoproterenol, in the presence of adenosine increased kinase activity but did not change glucose transport activity. Conversely, upon removal of adenosine, addition of Rs ligands such as isoproterenol, adrenocorticotropic hormone, or glucagon strongly inhibited transport (approximately 50%) and stimulated kinase activity. However, subsequent addition of phenylisopropyladenosine nearly restored transport activity without alteration of A-kinase activity. These data and additional kinetic experiments suggest that Rs-mediated glucose transport modulations are also independent of cAMP. The interchangeability of ligands for both Rs and Ri receptors in modulating transport activity suggests that these cAMP-independent effects are mediated by the stimulatory (Ns) and inhibitory (Ni) guanyl nucleotide-binding regulatory proteins of adenylate cyclase. All Rs-and Ri-induced changes in transport activity occurred without a change in glucose transporter distribution, as assessed by D-glucose-inhibitable cytochalasin B binding, suggesting that Rs and Ri ligands modulate the intrinsic activity of the glucose transporter present in the plasma membrane.

3-O-Methylglucose↗
PubMed1987

Doppler ultrasound in the assessment of severity of coarctation of the aorta and interruption of the aortic arch.

Doppler ultrasound was used to investigate 48 infants and children (age 2 days-16 years, weight 1.0-58 kg) with aortic arch abnormalities. In only 38 of the 42 with an important coarctation was an increased blood flow velocity from the distal arch demonstrated. In three with interruption of the aortic arch an increased velocity recorded from the region of the distal arch was thought to represent ductal flow. There was little difference between the peak to peak and instantaneous maximum gradients in the 20 patients with important coarctation in whom direct pressure measurements both proximal and distal to the obstruction were made at catheterisation. There were poor agreements between Doppler and measured peak to peak and instantaneous gradients in the 17 patients found to have both an increased velocity and important coarctation. It is concluded that although an increased blood flow velocity in the distal arch is usually demonstrated in coarctation this may not occur with severe obstruction. Furthermore, the maximum velocity is not related to the anatomical severity of the obstruction and the Doppler estimate of pressure drop in coarctation may not even reliably predict that measured at catheterisation.

Adolescent↗
PubMed1987

Ultrafiltration in diuretic-resistant cardiac failure.

Ultrafiltration was performed in 13 patients with diuretic-resistant cardiac failure. All patients had severely distressing peripheral edema and ultrafiltration was successfully completed in 12 patients, all of whom sustained symptomatic improvement. The volume of fluid removed ranged from 3.7 to 23 L, mean 11.8 L. Weight reduction produced by ultrafiltration continued over the following week, indicating an improved response to diuretic therapy. Ultrafiltration is an effective therapeutic intervention in the management of a small but well-defined group of patients with diuretic-resistant cardiac failure.

Aged↗
PubMed1986

Insulin-stimulated glucose transport in rat adipose cells. Modulation of transporter intrinsic activity by isoproterenol and adenosine.

The mechanism of modulation of insulin-stimulated glucose transport activity in isolated rat adipose cells by lipolytic and antilipolytic agents has been examined. We have measured glucose transport activity in intact cells with 3-O-methylglucose and in plasma membranes with D-glucose, and the concentration of glucose transporters in plasma membranes using a cytochalasin B binding assay. In intact cells, isoproterenol reduced insulin-stimulated transport activity by 60%. This effect was lost after cooling and washing the cells with homogenization buffer, and neither the concentration of glucose transporters nor transport activity in the plasma membranes differed from control. However, treatment of cells with KCN prior to homogenization preserved the isoproterenol effect through the fractionation procedure. Plasma membranes from these cells contained an unchanged number of transporters (31 +/- 7, mean +/- S.E., versus 31 +/- 4 pmol/mg of protein in controls) but transported glucose at a reduced rate (19 +/- 6 versus 48 +/- 9 pmol/mg of protein/s). Conversely, incubation of intact cells in the presence of adenosine stimulated plasma membrane glucose transport activity compared to that in the absence of adenosine (44 +/- 6 versus 36 +/- 6 pmol/mg of protein/s). Kinetic studies of isoproterenol-inhibited glucose transport in plasma membranes revealed a 60% decrease in Vmax (2900 +/- 350 versus 7200 +/- 1000 pmol/mg of protein/s) and a small increase in Km (15.1 +/- 1 versus 13.0 +/- 0.6 mM). These data indicate that modifications of glucose transport activity produced by lipolytic and antilipolytic agents in intact adipose cells can be fully retained in plasma membranes isolated under appropriate conditions. Furthermore, the effects of these agents occur through a modification of the glucose transporter intrinsic activity.

3-O-Methylglucose↗
PubMed1986

Biochemical and functional heterogeneity of rat adipocyte glucose transporters.

We have studied the biochemical mechanism of insulin action on glucose transport in the rat adipocyte. Plasma membranes and low-density microsomes were prepared by differential ultracentrifugation of basal and insulin-stimulated cells. The photochemical cross-linking agent hydroxysuccinimidyl-4-azidobenzoate was used to covalently bind [3H]cytochalasin B to the glucose transporter which migrated as a 45-50-kDa protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Isoelectric focusing of the eluted 40-55-kDa proteins revealed two peaks of D-glucose-inhibitable [3H]cytochalasin B radioactivity focusing at pH 6.4 and 5.6 when low-density microsomes were used as the starting material. In contrast, only one D-glucose inhibitable peak, focusing at pH 5.6, was found in plasma membranes. Pretreatment of the cells with insulin led to a marked redistribution of the pH 5.6 form of the glucose transporter from low-density microsomes to plasma membranes with no effect on the pH 6.4 form of the glucose transporter. Following isolation from the isoelectric focusing and sodium dodecyl sulfate-polyacrylamide gels, both glucose transporter isoforms were shown to cross-react with an antiserum raised against the purified human erythrocyte glucose transporter. Following incubation of [3H]cytochalasin B-labeled low-density microsomal and plasma membranes with neuraminidase, the pH 5.6 transporter isoform was shifted on isoelectric focusing to a more basic pH, while the pH 6.4 isoform was not affected. These data demonstrate that: there is a heterogeneity of glucose transporter species in the intracellular pool while the plasma membrane transporters are more uniform in structure. The pH 5.6 glucose transporter isoform is translocated by insulin from the low-density microsomes to the plasma membrane but the pH 6.4 isoform is not sensitive to insulin. Differential sensitivity of the glucose transporter isoforms to neuraminidase suggests that the heterogeneity is at least partially due to differences in glycosylation state.

Adipose Tissue↗