Effects of insulin on glucose transport and glucose transporters in rat heart.

The effect of insulin on glucose transport and glucose transporters was studied in perfused rat heart. Glucose transport was measured by the efflux of labelled 3-O-methylglucose from hearts preloaded with this hexose. Insulin stimulated 3-O-methylglucose transport by: (a) doubling the maximal velocity (Vmax); (b) decreasing the Kd from 6.9 to 2.7 mM; (c) increasing the Hill coefficient toward 3-O-methylglucose from 1.9 to 3.1; (d) increasing the efficiency of the transport process (k constant). Glucose transporters in enriched plasma and microsomal membranes from heart were quantified by the [3H]cytochalasin-B-binding assay. When added to normal hearts, insulin produced the following changes in the glucose transporters: (a) it increased the translocation of transporters from an intracellular pool to the plasma membranes; (b) it increased (from 1.6 to 2.7) the Hill coefficient of the transporters translocated into the plasma membranes toward cytochalasin B, suggesting the existence of a positive co-operativity among the transporters appearing in these membranes; (c) it increased the affinity of the transporters (and hence, possibly, of glucose) for cytochalasin B. The data provide evidence that the stimulatory effect of insulin on glucose transport may be due not to the sole translocation of intracellular glucose transporters to the plasma membrane, but to changes in the functional properties thereof.     

Identification of the D-glucose-inhibitable cytochalasin B binding site as the glucose transporter in rat diaphragm plasma and microsomal membranes

[3H]Cytochalasin B binding and its competitive inhibition by D-glucose have been used to identify, the glucose transporter in plasma and microsomal membranes prepared from intact rat diaphragm. Scatchard plot analysis of [3H]cytochalasin B binding yields a binding site with a dissociation constant of roughly 110 nM. Since the inhibition constant of cytochalasin B for D-glucose uptake by diaphragm plasma membranes is similar to this value, this site is identified as the glucose transporter. Plasma membranes prepared from diaphragms bind approx. 17 pmol of cytochalasin B/mg of membrane protein to the D-glucose-inhibitable site. If 280 nM (40000 microunits/ml) insulin is present during incubation, cytochalasin B binding is increased roughly 2-fold without alteration in the dissociation constant of this site. In addition, membranes in the microsomal fraction contain 21 pmol of D-glucose-inhibitable cytochalasin B binding sites/mg of membrane protein. In the presence of insulin during incubation the number of these sites in the microsomal fraction is decreased to 9 pmol/mg of membrane protein. These results suggest that rat diaphragm contain glucose transporters with characteristics identical to those observed for the rat adipose cell glucose transporter. In addition, insulin stimulates glucose transport in rat diaphragm through a translocation of functionally identical glucose transporters from an intracellular membrane pool to the plasma membrane without an alteration in the characteristics of these sites.     

Identification of the d-glucose-inhibitable cytochalasin B binding site as the glucose transporter in rat diaphragm plasma and microsomal membranes

[³H]Cytochalasin B binding and its competitive inhibition by d-glucose have been used to identify the glucose transporter in plasma and microsomal membranes prepared from intact rat diaphragm. Scatchard plot analysis of [³H]cytochalasin B binding yields a binding site with a dissociation constant of roughly 110 nM. Since the inhibition constant of cytochalasin B for d-glucose uptake by diaphragm plasma membranes is similar to this value, this site is identified as the glucose transporter. Plasma membranes prepared from diaphragms bind approx. 17 pmol of cytochalasin B/mg of membrane protein to the d-glucose-inhibitable site. If 280 nM (40 000 μunits/ml) insulin is present during incubation, cytochalasin B binding is increased roughly 2-fold without alteration in the dissociation constant of this site. In addition, membranes in the microsomal fraction contain 21 pmol of d-glucose-inhibitable cytochalasin B binding sites/mg of membrane protein. In the presence of insulin during incubation the number of these sites in the microsomal fraction is decreased to 9 pmol/mg of membrane protein. These results suggest that rat diaphragm contain glucose transporters with characteristics identical to those observed for the rat adipose cell glucose transporter. In addition, insulin stimulates glucose transport in rat diaphragm through a translocation of functionally identical glucose transporters from an intracellular membrane pool to the plasma membrane without an alteration in the characteristics of these sites.     

Identification of an intracellular pool of glucose transporters from basal and insulin-stimulated rat skeletal muscle

The purpose of this study was to simultaneously isolate skeletal muscle plasma and microsomal membranes from the hind limbs of male Sprague-Dawley rats perfused either in the absence or presence of 20 milliunits/ml insulin and to determine the effect of insulin on the number and distribution of glucose transporters in these membrane fractions. Insulin increased hind limb glucose uptake greater than 3-fold (2.4 +/- 0.7 versus 9.2 +/- 1.0 mumol/g x h, p less than 0.001). Plasma membrane glucose transporter number, measured by cytochalasin B binding, increased 2-fold (9.1 +/- 1.0 to 20.4 +/- 3.1 pmol/mg protein, p less than 0.005) in insulin-stimulated muscle while microsomal membrane transporters decreased significantly (14.8 +/- 1.6 to 9.8 +/- 1.4 pmol/mg protein, p less than 0.05). No change in the dissociation constant (Kd approximately 120 nm) was observed. K+-stimulated-p-nitrophenol phosphatase, 5'-nucleotidase, and galactosyltransferase specific activity, enrichment, and recovery in the plasma and microsomal membrane fractions were not altered by insulin treatment. Western blot analysis using the monoclonal antibody mAb 1F8 (specific for the insulin-regulatable glucose transporter) demonstrated increased glucose transporter densities in plasma membranes from insulin-treated hind limb skeletal muscle compared with untreated tissues, while microsomal membranes from the insulin-treated hind limb skeletal muscle had a concomitant decrease in transporter density. We conclude that the increase in plasma membrane glucose transporters explains, at least in part, the increase in glucose uptake associated with insulin stimulation of hind limb skeletal muscle. Our data further suggest that these recruited transporters originate from an intracellular microsomal pool, consistent with the translocation hypothesis.     

Potential mechanism of insulin action on glucose transport in the isolated rat diaphragm. Apparent translocation of intracellular transport units to the plasma membrane

[3H]Cytochalasin B binding and its competitive inhibition by D-glucose have been used to quantitate the number of functional glucose transport units in plasma and microsomal membranes prepared from intact rat diaphragm. In a series of three experiments, plasma membranes prepared from diaphragms which have not been incubated with insulin bind approximately 16 pmol of cytochalasin B/mg of membrane protein to the D-glucose-inhibitable binding site. If 280 nM (40,000 microunits/ml) insulin is present during the incubation, cytochalasin B binding to the plasma membranes is increased approximately 2-fold without alteration in the dissociation constant of this site. Membranes in the microsomal fraction prepared from diaphragms which have been incubated for 30 min in the absence of insulin contain 21 pmol of D-glucose-inhibitable cytochalasin B binding sites/mg of membrane protein. However, in the presence of insulin during the incubation period, the number of these sites in the microsomal fraction is decreased to 12 pmol/mg of membrane protein. These results suggest that insulin stimulates glucose transport in the isolated rat diaphragm primarily through a translocation of functional glucose transport units from an intracellular membrane pool to the plasma membrane. These results are similar to the results observed in rat adipose cells (Cushman, S. W., and Wardzala, L. J. (1980) J. Biol. Chem. 255, 4758-4762) and suggest that this mechanism of insulin-stimulated glucose transport activity may be general to other cell types.     

Insulin stimulates glucose transport in isolated human adipose cells through a translocation of intracellular glucose transporters to the plasma membrane: a preliminary report

Insulin's effect on glucose transport activity and the subcellular distribution of glucose transporters have been examined in isolated human abdominal adipose cells, by measuring 3-O-methylglucose transport and specific D-glucose-inhibitable cytochalasin B binding to plasma membranes and low-density microsomes, respectively. Insulin appears to stimulate glucose transport in isolated human adipose cell through the translocation of glucose transporters from a large intracellular pool to the plasma membrane as initially postulated for rat adipose and muscle cells.     

Mechanism for markedly hyperresponsive insulin-stimulated glucose transport activity in adipose cells from insulin-treated streptozotocin diabetic rats. Evidence for increased glucose transporter intrinsic activity

The effects of insulin therapy in streptozotocin diabetic rats on the glucose transport response to insulin in adipose cells have been examined. At sequential intervals during subcutaneous insulin infusion, isolated cells were prepared and incubated with or without insulin, and 3-O-methylglucose transport was measured. Insulin treatment not only reversed the insulin-resistant glucose transport associated with diabetes, but resulted in a progressive hyperresponsiveness, peaking with a 3-fold overshoot at 7-8 days (12.1 +/- 0.3 versus 3.4 +/- 0.1 fmol/cell/min, mean +/- S.E.) and remaining elevated for more than 3 weeks. During the peak overshoot, glucose transporters in subcellular membrane fractions were assessed by cytochalasin B binding. Insulin therapy restored glucose transporter concentration in the plasma membranes of insulin-stimulated cells from a 40% depleted level previously reported in the diabetic state to approximately 35% greater than control (38 +/- 4 versus 28 +/- 2 pmol/mg of membrane protein). Glucose transporter concentration in the low-density microsomes from basal cells was also restored from an approximately 45% depleted level back to normal (50 +/- 4 versus 50 +/- 6 pmol/mg of membrane protein), whereas total intracellular glucose transporters were further increased due to an approximately 2-fold increase in low-density microsomal membrane protein. However, these increases remained markedly less than the enhancement of insulin-stimulated glucose transport activity in the intact cell. Thus, insulin treatment of diabetic rats produces a marked and sustained hyperresponsive insulin-stimulated glucose transport activity in the adipose cell with little more than a restoration to the non-diabetic control level of glucose transporter translocation. Because this enhanced glucose transport activity occurs through an increase in Vmax, insulin therapy appears to be associated with a marked increase in glucose transporter intrinsic activity.     

Cycloheximide decreases glucose transporters in rat adipocyte plasma membranes without affecting insulin-stimulated glucose transport.

This study examines the relationship between insulin-stimulated glucose transport and insulin-induced translocation of glucose transporters in isolated rat adipocytes. Adipose cells were incubated with or without cycloheximide, a potent inhibitor of protein synthesis, for 60 min and then for an additional 30 min with or without insulin. After the incubation we measured 3-O-methylglucose transport in the adipose cells, and subcellular membrane fractions were prepared. The numbers of glucose transporters in the various membrane fractions were determined by the cytochalasin B binding assay. Basal and insulin-stimulated 3-O-methylglucose uptakes were not affected by cycloheximide. Furthermore, cycloheximide affected neither Vmax. nor Km of insulin-stimulated 3-O-methylglucose transport. In contrast, the number of glucose transporters in plasma membranes derived from cells preincubated with cycloheximide and insulin was markedly decreased compared with those from cells incubated with insulin alone (10.5 +/- 0.8 and 22.2 +/- 1.8 pmol/mg of protein respectively; P less than 0.005). The number of glucose transporters in cells incubated with cycloheximide alone was not significantly different compared with control cells. SDS/polyacrylamide-gel-electrophoretic analysis of [3H]cytochalasin-B-photolabelled plasma-membrane fractions revealed that cycloheximide decreases the amount of labelled glucose transporters in insulin-stimulated membranes. However, the apparent molecular mass of the protein was not changed by cycloheximide treatment. The effect of cycloheximide on the two-dimensional electrophoretic profile of the glucose transporter in insulin-stimulated low-density microsomal membranes revealed a decrease in the pI-6.4 glucose-transporter isoform, whereas the insulin-translocatable isoform (pI 5.6) was decreased. Thus the observed discrepancy between insulin-stimulated glucose transport and insulin-induced translocation of glucose transporters strongly suggests that a still unknown protein-synthesis-dependent mechanism is involved in insulin activation of glucose transport.     

Insulin-stimulated translocation of glucose transporters in the isolated rat adipose cells: characterization of subcellular fractions

Insulin stimulates glucose transport in rat adipose cells through the translocation of glucose transporters from an intracellular pool to the plasma membrane. A detailed characterization of the morphology, protein composition and marker enzyme content of subcellular fractions of these cells, prepared by differential ultracentrifugation, and of the distribution of glucose transporters among these fractions is now described. Glucose transporters were measured using specific D-glucose-inhibitable [3H]cytochalasin B binding. In the basal state, roughly 90% of the cells' glucose transporters are associated with a low-density microsomal, Golgi marker enzyme-enriched membrane fraction. However, the distributions of glucose transporters and Golgi marker enzyme activities over all fractions are clearly distinct. Incubation of intact cells with insulin increases the number of glucose transporters in the plasma membrane fraction 4-5 fold and correspondingly decreases the intracellular pool, without influencing any other characteristics of the subcellular fractions examined or the estimated total number of glucose transporters (3.7 X 10(6)/cell). Insulin does not influence the Kd of the glucose transporters in the plasma membrane fraction for cytochalasin B binding (98 nM), but lowers that in the intracellular pool (from 141 to 93 nM). The calculated turnover numbers of the glucose transporters in the plasma membrane vesicles from basal and insulin-stimulated cells are similar (15 X 10(3) mol of glucose/min per mol of transporters at 37 degrees C), whereas insulin appears to increase the turnover number in the plasma membrane of intact cells roughly 4-fold. These results suggest that (1) the intracellular pool of glucose transporters may comprise a specialized membrane species, (2) intracellular glucose transporters may undergo conformational changes during their cycling to the plasma membrane in response to insulin, and (3) the translocation of glucose transporters may represent only one component in the mechanism through which insulin regulates glucose transport in the intact cell.     

Effect of insulin on glucose transporter distribution in white fat cells from hypophysectomized rats

Glucose transport in white fat cells from hypophysectomized rats is increased and unresponsive to insulin. The goal of this study was to explain this observation. The number of glucose transporters, as determined by D-glucose-inhibitible cytochalasin B binding, in the plasma membranes from fat cells of hypophysectomized rats is: (1) elevated, (2) not increased by insulin, and (3) the same as in plasma membranes from insulin-stimulated fat cells of control rats. In microsomal membranes from fat cells of hypophysectomized rats the number of glucose transporters is: (1) smaller than in basal and insulin-stimulated fat cells from control rats, and (2) not changed by insulin.     

Proposed mechanism for increased insulin-mediated glucose transport in adipose cells from young, obese Zucker rats. Large intracellular pool of glucose transporters

The mechanism for hyperresponsive insulin-mediated glucose transport in adipose cells from 30-day-old obese Zucker rats was examined. Glucose transport was assayed by measuring 3-O-methylglucose transport, and the concentration of glucose transporters was estimated by measuring specific D-glucose-inhibitable cytochalasin B binding. Insulin increased glucose transport activity by approximately 17 fmol/cell/min in cells from obese rats compared to 3 fmol/cell/min in lean littermates. Insulin increased the concentration of glucose transporters in the plasma membrane fraction by about 15 pmol/mg of membrane protein in both groups. The insulin-mediated decrease in the concentration of transporters in the low-density microsomal fraction was 30 pmol/mg of membrane protein for the obese rats compared to 15 pmol/mg of membrane protein for the lean controls. An estimated number of glucose transporters was calculated using membrane protein and enzyme recoveries for each group. Insulin increased the number of transporters in the plasma membrane by 3 X 10(6) sites/cell for the obese rats and only 0.6 X 10(6) sites/cell for the lean controls. In addition, insulin decreased the number of transporters/cell in the intracellular membrane pool by approximately 4 X 10(6) sites/cell for the obese rats and 0.9 X 10(6) sites/cells for the lean rats. The total number of transporters/cell was about 7 X 10(6) sites/cell for the obese animals and 1.6 X 10(6) sites/cell for the lean controls. In the basal state, more than 80% of these transporters were located in the intracellular pool for both the lean and obese rats. Thus, the marked hyperresponsive insulin-mediated glucose transport observed in adipose cells from 30-day-old obese Zucker rats may be the consequence of a marked increase in the number of glucose transporters in the intracellular pool.     

Insulin-stimulated translocation of glucose transporters in the isolated rat adipose cells: Characterization of subcellular fractions

Insulin stimulates glucose transport in rat adipose cells through the translocation of glucose transporters from an intracellular pool to the plasma membrane. A detailed characterization of the morphology, protein composition and marker enzyme content of subcellular fractions of these cells, prepared by differential ultracentrifugation, and of the distribution of glucose transporters among these fractions is now described. Glucose transporters were measured using specific d-glucose-inhibitable [³H]cytochalasin B binding. In the basal state, roughly 90% of the cells' glucose transporters are associated with a low-density microsomal, Golgi marker enzyme-enriched membrane fraction. However, the distributions of glucose transporters and Golgi marker enzyme activities over all fractions are clearly distinct. Incubation of intact cells with insulin increases the number of glucose transporters in the plasma membrane fraction 4–5-fold and correspondingly decreases the intracellular pool, without influencing any other characteristics of the subcellular fractions examined or the estimated total number of glucose transporters (3.7·10⁶/cell). Insulin does not influence the K_d of the glucose transporters in the plasma membrane fraction for cytochalasin B binding (98 nM), but lowers that in the intracellular pool (from 141 to 93 nM). The calculated turnover numbers of the glucose transporters in the plasma membrane vesicles from basal and insulin-stimulated cells are similar (15·10³ mol of glucose/min per mol of transporters at 37°C), whereas insulin appears to increase the turnover number in the plasma membrane of intact cells roughly 4-fold. These results suggest that (1) the intracellular pool of glucose transporters may comprise a specialized membrane species, (2) intracellular glucose transporters may undergo conformational changes during their cycling to the plasma membrane in response to insulin, and (3) the translocation of glucose transporters may represent only one component in the mechanism through which insulin regulates glucose transport in the intact cell.     

Insulin-mediated translocation of glucose transporters from intracellular membranes to plasma membranes: sole mechanism of stimulation of glucose transport in L6 muscle cells

Plasma membranes and light microsomes were isolated from fused L6 muscle cells. Pre-treatment of cells with insulin did not affect marker enzyme or protein distribution in isolated membranes. The number of glucose transporters in the isolated membranes was calculated from the D-glucose-protectable binding of [3H]cytochalasin B. Glucose transporter number was higher in plasma membranes and lower in intracellular membranes derived from insulin-treated cells than in the corresponding fractions from untreated cells. The net increase in glucose transporters in plasma membranes was identical to the net decrease in glucose transporters in light microsomes (2 pmol/1.23 x 10(8) cells). The fold increase in glucose transporter number/mg protein in plasma membranes (2-fold) was similar to the fold increase in glucose transport caused by insulin. This suggests that recruitment of glucose transporters from intracellular membranes to the plasma membrane is the major mechanism of stimulation of hexose transport in L6 muscle cells. This is the first report of isolation of the two insulin-sensitive membrane elements from a cell line, and the results indicate that, in contrast to rat adipocytes, there is not change in the intrinsic activity of the transporters in response to insulin.     

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.     

Reconstitution of the glucose transporter from bovine heart

Reconstitution of the glucose transporter from heart should be useful as an assay in its purification and in the study of its regulation. We have prepared plasma membranes from bovine heart which display D-glucose reversible binding of cytochalasin B (33 pmol sites/mg protein; Kd = 0.2 muM). The membrane proteins were reconstituted into liposomes by the freeze-thaw procedure. Reconstituted liposomes showed D-glucose transport activity which was stereospecific, saturable and inhibited by cytochalasin B, phloretin, and mercuric chloride. Compared to membrane proteins reconstituted directly, proteins obtained by dispersal of the membranes with low concentrations of cholate or by cholate solubilization showed 1.2- or 2.3-fold higher specific activities for reconstituted transport, respectively. SDS-polyacrylamide gel electrophoresis followed by electrophoretic protein transfer and labeling with antisera prepared against the human erythrocyte transporter identified a single band of about 45 kDa in membranes from both dog and bovine hearts, a size similar to that reported for a number of other glucose transporters in various animals and tissues.     

Glucose transporters in chromaffin cells: subcellular distribution and characterization

The glucose transporter was identified and characterized by cytochalasin B binding in subcellular membrane fractions of chromaffin tissue. The binding was saturable with Kd of about 0.3 microM for each subcellular fraction. The Bmax capacity was 12-16 pmol/mg protein for enriched plasma membrane fractions, 6.3 pmol/mg protein for microsomal membrane preparations and 5.4 pmol/mg protein for chromaffin granule membranes. Irreversible photoaffinity labelling of the glucose-protectable binding sites with [3H]cytochalasin B followed by solubilization and polyacrylamide gel electrophoresis from enriched plasma membrane preparations demonstrated the presence of three molecular species: 97 +/- 10, 51.5 +/- 6 and 30 +/- 4 kDa. The chromaffin granule membranes showed only a molecular species of 80 +/- 10 kDa.     

Cold exposure increases glucose utilization and glucose transporter expression in brown adipose tissue.

When rats were exposed to a cold environment (4 degrees C) for 10 days, tissue glucose utilization was increased in brown adipose tissue (BAT), a tissue specified for non-shivering thermogenesis, but not in skeletal muscle. Cold exposure also caused an increase in the amount of GLUT4, an isoform of glucose transporters expressed in insulin-sensitive tissues, in parallel with an increased cellular level of GLUT4 mRNA. In contrast to BAT, no significant effect of cold exposure was found in skeletal muscle. The results suggest the cold-induced increase in glucose utilization by BAT is attributable, at least in part, to the increased expression of GLUT4.     

Biochemical and functional characterization of the rat liver glucose-transport system. Comparisons with the adipocyte glucose-transport system.

The properties of the glucose-transport systems in rat adipocytes and hepatocytes were compared in cells prepared from the same animals. Hormones and other agents which cause a large stimulation of 3-O-methylglucose transport in adipocytes were without acute effect in hepatocytes. Hepatocytes displayed a lower affinity for 3-O-methylglucose (20 mM) and alternative substrates than adipocytes (6 mM), whereas inhibitor affinities were similar in both cell types. The concentration and distribution of glucose transporters were determined by Scatchard analysis of D-glucose-inhibitable [3H]cytochalasin B binding to subcellular fractions. In liver, most of the transporters were located in the plasma membrane (42 +/- 5 pmol/mg of protein) with a small amount (4 +/- 3 pmol/mg) in the low-density microsomal fraction ('microsomes'), the reverse of the situation in adipocytes. Glucose transporters were covalently labelled with [3H]cytochalasin B by using the photochemical cross-linking agent hydroxysuccinimidyl-4-azidobenzoate and analysed by SDS/polyacrylamide-gel electrophoresis. A single D-glucose-inhibitable peak with a molecular mass of 40-50 kDa was seen in both plasma membrane and low-density microsomes. This peak was further characterized by isoelectric focusing and revealed a single peak of specific [3H]cytochalasin B binding at pI 6.05 in both low-density microsomes and plasma membrane, compared with peaks at pI 6.4 and 5.6 in adipocyte membranes. In summary: the glucose-transport system in hepatocytes has a lower affinity and higher capacity than that in adipocytes, and is also not accurately modulated by insulin; the subcellular distribution of glucose transporters in the liver suggests that few intracellular transporters would be available for translocation; the liver transporter has a molecular mass similar to that of the adipocyte transporter; the liver glucose transporter exists as a single charged form (pI 6.05), compared with the multiple forms in adipocytes. This difference in charge could reflect a functionally important difference in molecular structure between the two cell types.     

Proposed mechanism of insulin-resistant glucose transport in the isolated guinea pig adipocyte. Small intracellular pool of glucose transporters

A marked resistance to the stimulatory action of insulin on glucose metabolism has previously been shown in guinea pig, compared to rat, adipose tissue and isolated adipocytes. The mechanism of insulin resistance in isolated guinea pig adipocytes has, therefore, been examined by measuring 125I-insulin binding, the stimulatory effect of insulin on 3-0-methylglucose transport and on lipogenesis from [3-3H]glucose, the inhibitory effect of insulin on glucagon-stimulated glycerol release, and the translocation of glucose transporters in response to insulin. The translocation of glucose transporters was assessed by measuring the distribution of specific D-glucose-inhibitable [3H]cytochalasin B binding sites among the plasma, and high and low density microsomal membrane fractions prepared by differential centrifugation from basal and insulin-stimulated cells. At a glucose concentration (0.5 mM) where transport is thought to be rate-limiting for metabolism, insulin stimulates lipogenesis from 30 to 80 fmol/cell/90 min in guinea pig cells and from 25 to 380 fmol/cell/90 min in rat cells with half-maximal effects at approximately 100 pM in both cell types. Insulin similarly stimulates 3-O-methylglucose transport from 0.40 to 0.70 fmol/cell/min and from 0.24 to 3.60 fmol/cell/min in guinea pig and rat fat cells, respectively. Nevertheless, guinea pig cells bind more insulin per cell than rat cells, and insulin fully inhibits glucagon-stimulated glycerol release. In addition, the differences between guinea pig and rat cells in the stimulatory effect of insulin on lipogenesis and 3-O-methylglucose transport cannot be explained by the greater cell size of the former compared to the latter (0.18 and 0.09 micrograms of lipid/cell, respectively). However, the number of glucose transporters in the low density microsomal membrane fraction prepared from basal guinea pig cells is markedly reduced compared to that from rat fat cells (12 and 70 pmol/mg of membrane protein, respectively) and the translocation of intracellular glucose transporters to the plasma membrane fraction in response to insulin is correspondingly reduced. These results suggest that guinea pig adipocytes are markedly resistant to the stimulatory action of insulin on glucose transport and that this resistance is the consequence of a relative depletion in the number of intracellular glucose transporters.     

Differential regulation of glucose transporter gene expression in adipose tissue or septic rats.

To understand the molecular mechanisms responsible for the sepsis-induced enhanced glucose uptake, we have examined the levels of GLUT4 and GLUT1 mRNA and protein in the adipose tissue of septic animals. Rats were challenged with a nonlethal septic insult where euglycemia was maintained and hexose uptake in adipose tissue was markedly elevated. Northern blot analysis of total RNA isolated from epididymal fat pads indicated differential regulation of the mRNA content for the two transporters: GLUT1 mRNA was increased 2.6 to 4.6-fold, while GLUT4 mRNA was decreased by 2.5 to 2.9-fold. Despite the difference in mRNA levels, both GLUT1 and GLUT4 protein were down regulated in plasma membranes (40% and 25%, respectively) and microsomal membranes (42% and 25%, respectively) of the septic animals. The increased glucose uptake cannot be explained by the membrane content of GLUT1 and GLUT4 protein. Thus, during hypermetabolic sepsis, increased glucose utilization by adipose tissue is dependent on alternative processes.