Methylglucose Transport Research Articles

Insulin acts via mitogen-activated protein kinase phosphorylation in rabbit blastocysts

The addition of insulin during in vitro culture has beneficial effects on rabbit preimplantation embryos leading to increased cell proliferation and reduced apoptosis. We have previously described the expression of the insulin receptor (IR) and the insulin-responsive glucose transporters (GLUT) 4 and 8 in rabbit preimplantation embryos. However, the effects of insulin on IR signaling and glucose metabolism have not been investigated in rabbit embryos. In the present study, the effects of 170 nM insulin on IR, GLUT4 and GLUT8 mRNA levels, Akt and Erk phosphorylation, GLUT4 translocation and methyl glucose transport were studied in cultured day 3 to day 6 rabbit embryos. Insulin stimulated phosphorylation of the mitogen-activated protein kinase (MAPK) Erk1/2 and levels of IR and GLUT4 mRNA, but not phosphorylation of the phosphatidylinositol 3-kinase-dependent protein kinase, Akt, GLUT8 mRNA levels, glucose uptake or GLUT4 translocation. Activation of the MAPK signaling pathway in the absence of GLUT4 translocation and of a glucose transport response suggest that in the rabbit preimplantation embryo insulin is acting as a growth factor rather than a component of glucose homeostatic control.

Correlation between local glucose transporter densities and local 3- O-methylglucose transport in rat brain

The present study addresses the question whether local glucose transport kinetics are correlated with local glucose transporter densities in the brain. In 47 brain structures the local rate constants for 3- O-[ 14C]methylglucose (3- O-MG) transport, K 1 and k 2, were quantified, and local glucose Glut1 and Glut3 transporter densities were determined by immuno-autoradiographic methods. Statistically significant correlations were found between the rate constants for glucose transport and the transporter densities. The correlations were tighter for Glut1 than for Glut3. Inasmuch as 3- O-MG is transported by the same transporter as glucose, these results indicate that the local densities of glucose transporters determine local glucose transport rates in the brain.

Inhibition of IRS-1 Phosphorylation and the Alterations of GLUT4 in Isolated Adipocytes from Cachectic Tumor-Bearing Rats

Cellular and molecular mechanisms of insulin resistance in isolated adipocytes from methylcholanthrene-induced sarcoma-bearing rats were investigated by measuring 3-O-[14C]methyl glucose transport activity, glucose transporter-4 (GLUT4) protein in both plasma membrane and low-density microsomes, and insulin-stimulated tyrosine phosphorylation of the insulin receptor (IR) and insulin receptor substrate-1 (IRS-1). Compared to both pair-fed and freely fed controls, tumor-bearing rats (TBR) had a decreased insulin-stimulated glucose transport activity with a lowerVmaxand a higher EC50. GLUT4 protein in low-density microsomes from adipocytes maintained at the basal state was less in TBR than in controls. In insulin-stimulated adipocytes, GLUT4 protein in plasma membranes was also less in tumor-bearing rats than in controls. Insulin-induced tyrosine phosphorylation of IRS-1 was less in TBR than controls, but that of the IR was similar among the three groups. These data suggest that the insulin resistance seen in adipose cells of these tumor-bearing rats was caused in part by a decreased amount of GLUT4 protein in both basal and insulin-stimulated states resulting from the selective inhibition of insulin-stimulated phosphorylation of IRS-1.

Insulin resistance and the alterations of glucose transporter-4 in adipose cells from cachectic tumor-bearing rats.

Insulin resistance may play an important role in cancer cachexia; however, its mechanisms remain to be clarified. Cellular mechanisms of insulin resistance in tumor-bearing rats (TBR) were investigated in isolated adipose cells by measuring 3-O-[14C]methyl glucose transport activity and glucose transporter-4 (GLUT4) protein in low-density microsomes at a basal state and in the plasma membrane at an insulin-stimulated state. The insulin-stimulated glucose transport activity in adipose cells from TBR was significantly lower than that of control rats (CTR) (0.51 +/- 0.25 and 2.27 +/- 0.11 fmol/cell/min, respectively). The amount of GLUT4 in low-density microsomes at a basal state and in plasma membrane at an insulin-stimulated state was less in TBR than in CTR. These data suggest that the insulin resistance seen in the adipose cells of these tumor-bearing rats was caused in part by both a decreased amount of GLUT4 protein in a basal state and a decreased translocation of GLUT4 in response to insulin stimulation.

The effect of lesions of the sympathoadrenal system on training induced adaptations in adipocytes and pancreatic islets in rats.

Physical training increases insulin stimulated glucose uptake in adipocytes and decreases insulin secretion from pancreatic islets. The mechanism behind these adaptations is not known. Because in acute exercise adrenergic activity influences both adipocytes and pancreatic islets, the sympathetic nervous system was examined as the possible mediator. Rats were either adrenodemedullated or sham adrenodemedullated and underwent either unilateral abdominal sympathectomy or were sham sympathectomized. Resting plasma adrenaline concentration in adrenodemedullated rats was 32% of the concentration in sham adrenodemedullated rats (P < 0.0001) and muscle noradrenaline content in sympathectomized leg was 9% of content in sham sympathectomized leg (P < 0.0001). After operations rats were either swim trained for 10 weeks or remained sedentary. Insulin stimulated 3-O-[14C]methylglucose transport was measured in adipocytes from epididymal fat pads, and insulin secretion and glucose metabolism were measured in glucose stimulated pancreatic islets. Training increased insulin stimulated glucose transport in adipocytes (P < 0.0001) and decreased their size (P < 0.0001), but neither adrenodemedullation nor sympathetic denervation affected these parameters significantly. Training decreased insulin secretion (P < 0.01) and increased glucose oxidation (P = 0.02) and utilization (P = 0.08) in pancreatic islets, but none of these parameters was affected significantly by adrenodemedullation. It is concluded that adrenergic activity is not important for the training induced decrease in size and increase in insulin stimulated glucose transport of adipocytes. Neither is an intact adrenal medulla necessary for training-induced adaptations in pancreatic beta cell function. Finally, in response to training, beta cell insulin secretion and glucose metabolism changed in opposite directions.

Accelerated net efflux of [formula omitted] from rat adipocytes: a reevaluation

In a study of 3-O- methylglucose transport in insulin-stimulated rat adipocytes (catalyzed primarily by the GLUT4 isoform), it was reported that at 37°C the K m and V max were 2.8-fold higher for net efflux than for equilibrium exchange (Vinten, J. (1984) Biochim. Biophys. Acta 772, 244–250). Because of its implications for the relative sizes of steps in the transport cycle, we reinvestigated this phenomenon. Accelerated net efflux was apparent when the extracellular methylglucose was diluted 26-fold but not when it was diluted 11-fold. When analyzed according to the one-site alternating conformation model, the data indicate about a 1.7-fold higher V max for efflux than for exchange, only about 40% of the difference reported previously. Together with other results in the literature, the accelerated net flux indicates that the conformational change of the loaded transporter from its outward-facing to its inward-facing form is likely the slowest step in the transport cycle, in contrast to the case for GLUT1. Experiments at 25°C indicate a lower degree of accelerated net flux than at 37°C. This is consistent with the above conformational change being the step with the lowest activation energy, as for GLUT1.

The effect of TGFα on intestinal solute transport

The effect of transforming growth α (TGFα) and epidermal growth factor (EGF) on 3-O- methylglucose transport was examined in vitro under short-circuited conditions in stripped rabbit jejunum. Mucosal EGF, 60 ng/ml, stimulated a significant increase in net 3-O- methylglucose transport ( J net 0.67 ± 0.15 vs. 0.90 ± 0.15 μ Eq/cm 2/ h ; P < 0.03; n = 6) due to an increased mucosal to serosal flux ( J ms 1.2 ± 0.2 vs. 1.5 ± 0.2 μ Eq/cm 2/ h ; P < 0.03). In contrast, TGFα, when applied to both mucosal and serosal surfaces at concentrations of either 60 ( n = 6) or 150 ( n = 9) ng/ml had no effect on either mucosal to serosal ( J ms ) or net transport ( J net ) of 3-O- methylglucose . TGFα did induce a significant increase in the serosal to mucosal flux ( J sm 60 ng/ml 0.44 ± 0.02 vs. 0.51 ± 0.03, 150 ng/ml 0.55 ± 0.03 vs. 0.64 ± 0.05 μ Eq/cm 2/ h ; P < 0.05). When brush border surface area was examined after exposure to either 60 ng/ml TGFα or saline vehicle for 2 h in in vivo isolated jejunal loops no significant difference was found (control 53 ± 1.9; n = 35 vs. TGFα 52 ± 1.9 μ m 2 ; n = 29). Bioactivity of transforming growth factor α was assessed by an gastric acid secretion bioassay and found to be intact. These data provide further evidence for separate and distinct functional roles for these peptides in some biological systems.

Postbinding insulin resistance around parturition in the isolated rat epitrochlearis muscle

To determine whether insulin resistance exists in maternal skeletal muscle during pregnancy and how it returns to normal during the postpartum period, 3-O-methyl[14C]-D-glucose transport and [125I]insulin-binding activities were measured in isolated rat epitrochlearis muscle. Maximally insulin-stimulated methylglucose transport activity was decreased on day 20 of pregnancy and on days 1 and 4 post partum; it returned to the nonpregnant level by day 9. The insulin-binding activity did not change significantly throughout pregnancy, increased on days 1 and 4 post partum, and returned to the nonpregnant level by day 9. There was no significant difference in insulin binding or insulin-stimulated methylglucose transport activity between lactating and nonlactating animals. These results suggest that insulin resistance caused by postbinding changes in epitrochlearis muscle develops during late pregnancy and continues at least until day 4 post partum. Lactation does not appear to have a significant effect on insulin resistance.

Postbinding insulin resistance around parturition in the isolated rat epitrochlearis muscle

To determine whether insulin resistance exists in maternal skeletal muscle during pregnancy and how it returns to normal during the postpartum period, 3-O-methyl[¹⁴C]-D-glucose transport and [¹²⁵I]insulin-binding activities were measured in isolated rat epitrochlearis muscle. Maximally insulin-stimulated methylglucose transport activity was decreased on day 20 of pregnancy and on days 1 and 4 post partum; it returned to the nonpregnant level by day 9. The insulin-binding activity did not change significantly throughout pregnancy, increased on days 1 and 4 post partum, and returned to the nonpregnant level by day 9. There was no significant difference in insulin binding or insulin-stimulated methylglucose transport activity between lactating and nonlactating animals. These results suggest that insulin resistance caused by postbinding changes in epitrochlearis muscle develops during late pregnancy and continues at least until day 4 post partum. Lactation does not appear to have a significant effect on insulin resistance.

Rabbit kidney proximal tubule cells in primary culture: Evaluation of the impact of expressed phenotype on cellular toxic response

A primary culture of rabbit kidney proximal tubule cells has been developed from highly purified and calibrated fragments of proximal tubules. Cells are grown without serum in various media. Minimum medium (MM) was composed of glucose-free Dulbecco's modified Eagle's medium plus Ham's F12 (1:1) fortified with sodium selenite plus hydrocortisone plus transferrin. The five types of culture medium used for this study were MM plus 15 m m-glucose with or without insulin (media Ins + G1 15 m m; Ins − G1 15 m m), MM plus 2.5 m m-glucose, and MM with or without insulin (Ins + G1 −; Ins − G1 −). Cellular phenotype is characterized by the assessment of (a) the specific activities of eight enzymatic markers, and (b) cellular functions such as DNA and protein synthesis, and methylglucose transport. This study emphasizes the importance of the effect of the composition of the culture medium on the quality of the phenotype expressed by proximal tubule cells in primary culture. In addition, a study involving the well-known nephrotoxic agent gentamicin has demonstrated the impact of the expressed phenotype on the onset and expression of the specific toxicity pattern: the best gentamicin-toxicity pattern is seen in a medium deprived of insulin and glucose.

Temperature dependence of glucose transport in erythrocytes from normal and alloxan-diabetic rats

Alloxan diabetes increased 3-O- methylglucose transport rates in rat red blood cells (RBC) at temperatures below 30°C and decreased them above 30°C. Preincubation of RBC from control rats with 20 mM glucose, 3-O- methylglucose , 2-deoxyglucose or xylose greatly elevated transport at 14°C by increasing V max . The effect was slight at 40°C. Preincubation with glucose or deoxyglucose alone caused a 50% depression of transport rates at 40°C as a result of a rise in the K m , which is similar to findings in cells from alloxan-diabetic rats. Measurement of intracellular glucose metabolites suggested inhibition of glycolysis in cells from diabetic rats and a positive correlation between the level of intracellular hexose monophosphates and transport inhibition. Membrane fatty-acid and cholesterol composition and membrane lipid-ordering as monitored by electron paramagnetic resonance were not altered by alloxan diabetes. It is concluded that intracellular sugar and sugar metabolism alter the temperature dependence of glucose transport kinetics. Glucose metabolism can feed back to inhibit transport by increasing the transport K m at physiological temperatures only.

Fasting-mediated alteration studies in insulin action on lipolysis and lipogenesis in obese women.

The effects of fasting on insulin-induced antilipolysis and lipogenesis were investigated in vitro in isolated human fat cells of 11 obese females. Glycerol release and lipogenesis were determined simultaneously in the same test tube and related to methylglucose transport and specific insulin binding. Insulin binding and sensitivity and the responsiveness (maximum effect) of insulin-induced antilipolysis were enhanced by fasting. The latter was strongly correlated with an enhancement in the lipolysis rate. The effects of fasting on antilipolysis were not dependent on the glucose concentration, unlike insulin-stimulated lipogenesis. At 1 mumol/l of glucose, where hexose transport is rate limiting, sensitivity and responsiveness of insulin-induced lipogenesis were inhibited by fasting. Similar results were obtained with methylglucose transport. At 1-10 mmol/l of glucose, where hexose metabolism is rate limiting, insulin stimulated lipogenesis before fasting but was totally ineffective after fasting. In conclusion, fasting induces multiple alterations in insulin action on lipolysis and lipogenesis in adipocytes. Antilipolysis is enhanced because of stimulation at the receptor and postreceptor levels, which may be associated with an enhanced rate of lipolysis. Fasting inhibits the lipogenic effect of insulin due to postreceptor changes involving both transport and metabolism of glucose, making lipogenesis unresponsive to insulin at physiological glucose concentrations.

3-O-methylglucose transport in soleus muscle of bacteremic rats.

Basal and insulin-stimulated soleus muscle 3-O-[14C]methylglucose ([14C]-3-O-MG) transport was studied in vitro and in vivo during bacteremia in rats. Fasted rats were injected with Escherichia coli to produce bacteremia (B), and controls (C) received saline. In vitro studies using soleus muscles were carried out 8 or 12 h after bacterial injection, and transport was measured using the rate coefficient (lambda = min-1). Although insulin-stimulated (10 mU/ml) [14C]-3-O-MG transport was decreased in 12-h bacteremic rat muscles (lambda B = 0.041 +/- 0.003; lambda C = 0.055 +/- 0.002), the basal [14C]-3-O-MG transport rate coefficient was elevated (lambda B = 0.027 +/- 0.004; lambda C = 0.019 +/- 0.001). For in vivo studies, [14C]-3-O-MG with or without insulin was injected into rats 10-40 min prior to removing soleus muscles at 12 h postbacterial or postsaline injection. Transport was measured as the ratio of [14C]-3-O-MGintracell/[14C]-3-O-MGextracell. Basal ratios were not different and muscles from both control and bacteremic rats responded comparably to insulin with increased [14C]-3-O-MG transport during the initial 30 min. At 35-40 min postinsulin injection there was a further stimulation of [14C]-3-O-MG transport in control but not in 12-h bacteremic rat muscles. The changes in [14C]-3-O-MG transport observed in vitro and in vivo after 12 h of bacteremia may be due to circulating mediators and/or changes in membrane function.

Metabolic basis for the diabetogenic action of growth hormone in the obese (ob/ob) mouse.

The ob/ob mouse responds predictably to chronic treatment with large doses of pituitary GH with marked hyperglycemia and decreased glucose tolerance. The purpose of the present study was to characterize the metabolic alterations produced by GH that lead to this diabetogenic response in the ob/ob mouse in order to determine whether this animal might serve as a useful model for the study of the cellular mechanisms involved in the diabetogenic action of GH. Female ob/ob mice were treated sc for 3 days with either saline or 200 micrograms/day S-carboxymethylated human GH (RCM-hGH), a diabetogenic GH derivative lacking significant growth-promoting or insulin-like activities. Six hours before the start of the experiment, the animals were given a sc injection of 2 micrograms dexamethasone and deprived of food. RCM-hGH treatment produced marked increases in fasting blood glucose and plasma insulin concentrations, but had no effect on plasma glucagon or serum insulin-like growth factor I levels. It had no effect on liver glycogen level or in vitro hepatic glucose production in the absence or presence of pyruvate and lactate added to the incubation medium. By contrast, the in vitro stimulatory effects of insulin on [14C] glucose oxidation by isolated soleus muscle or segments of parametrial fat were greatly attenuated by RCM-hGH treatment, without changes in rates of basal glucose oxidation. This change in peripheral tissue responsiveness to insulin does not appear to involve glucose transport, since the in vitro stimulation by insulin of 3-O-[14C]methylglucose transport into isolated diaphragm muscle was not altered by RCM-hGH treatment. Moreover, the RCM-hGH-induced reduction in adipose tissue responsiveness to insulin does not appear to be mediated by a reduction in insulin binding, since [125I]iodoinsulin binding to adipocytes isolated from RCM-hGH-treated mice was similar to that to cells from saline-treated animals. Interestingly, the reduction in responsiveness to insulin seen with segments of adipose tissue from RCM-hGH-treated animals was not found with isolated adipocytes prepared from such tissue by collagenase digestion. These results suggest that the hyperglycemia and glucose intolerance produced in ob/ob mice by chronic GH treatment result primarily from increased peripheral tissue insulin resistance. Therefore, the ob/ob mouse provides a useful model to elucidate the cellular mechanism(s) of this aspect of the diabetogenic action of GH.

Effects of insulin on hexose transport across blood-brain barrier in normoglycemia.

The effects of insulin on 3-O-[14C]methylglucose transport across the blood-brain barrier (BBB) were studied in conscious rats under steady-state normoglycemic conditions. The [14C]methylglucose was infused intravenously at a constant rate, and animals were killed at various times between 5 and 30 min after the initiation of the infusion. The time course of the arterial plasma concentration of [14C]methylglucose was determined in timed arterial blood samples taken during the infusion. Local cerebral tissue concentrations of [14C]methylglucose at the time of killing were determined by quantitative autoradiography of brain sections. The rate constants for inward and outward transport of [14C]methylglucose across the BBB, K1, and k2, respectively, were estimated by a least-squares, best-fit of a kinetic equation to the measured time courses of plasma and tissue concentrations. K1 and k2 were reduced by an average of 24 and 31%, respectively, in gray matter and 7 and 16% in white matter from values estimated similarly in normal insulinemic control rats. The equilibrium distribution ratio, K1/k2, for [14C]methylglucose in brain increased by approximately 10-11% in the hyperinsulinemic animals. Because 3-O-[14C]methylglucose shares the same carrier that transports glucose and other hexoses across the BBB, these results suggest that hyperinsulinemia decreases the rate constants for transport but increases the distribution space for hexoses in brain. These effects are, however, quite small and are probably minor or negligible when compared with the major effects of insulin in other tissues.

Modulation of basal glucose transporter Km in the adipocyte by insulin and other factors.

We have previously described experimental conditions where basal methylglucose transport in adipocytes exhibited an apparent Km of approximately 35 mM. Under those conditions insulin stimulated transport predominantly by decreasing the transport Km (Whitesell, R. R., and Abumrad, N. A. (1985) J. Biol. Chem. 260, 2894-2899). Our findings were in contrast with earlier reports that the Km of basal glucose transport was low (3-5 mM) and similar to that of transport in insulin-treated cells. In this study we have investigated the effect of different experimental conditions on the kinetics of basal glucose transport in adipocytes. When transport was assayed at 37 degrees C, cell agitation for 10 min prior to the transport assay decreased the basal Km from 35 to 12 mM. Deprivation of metabolic substrate produced a further reduction down to 2 mM. Refeeding starved cells with 1 mM glucose returned the Km back up to 12 mM in agitated cells and to 40 mM in stabilized cells. The effects of agitation to lower and of glucose to raise the basal Km were prevented by preincubating cells with dinitrophenol. Cell agitation or substrate lack did not alter the Vmax of basal transport and were without effect on both Km and Vmax in insulin-treated cells. The temperature dependencies of the kinetics of basal and stimulated transport were studied. A decrease in the assay temperature from 37 to 23 degrees C caused both basal Km and Vmax to drop proportionately from 25 to 5 mM, and 13 to 3.6 nmol/(microliter X min), respectively. In insulin-stimulated cells, only the Vmax was decreased (Km went from 3.5 to 3 mM, Vmax from 45 to 17 nmol/(microliter X min]. The results support the concept that experimental conditions can produce large changes in the Km of basal glucose transporters. Furthermore they explain why, under certain assay conditions (with temperatures around 23 degrees C or with deprivation of metabolic substrate), the effect of insulin on transport Km is not observed. Our data also suggest that basal transport characteristics do not persist in insulin-treated cells. We would propose that one of the actions of insulin (in addition to raising Vmax) is to change the characteristics of basal transporters by overriding metabolic factors which keep the Km high. Alternatively, insulin could cause the disappearance of basal transporters as new and different ones are recruited from intracellular stores.

Effect of puromycin on sugar transport in isolated rat adipocytes

This study was performed to determine whether puromycin effects sugar transport into isolated rat adipocytes. Time-course and kinetic studies demonstrated that puromycin competitively inhibited 3-O- methylglucose transport. The data indicate that there is an acute effect of puromycin on sugar transport via competitive inhibition that is independent of the inhibition of protein synthesis.

The effect of catecholamines and adenosine deaminase on the glucose transport system in rat adipocytes

2-Deoxyglucose uptake (3 min) and 3-O- methylglucose transport (2 s) was measured in rat adipocytes preincubated with 5 μM epinephrine plus adenosine deaminase as described by Green (Green, A. (1983) FEBS Lett. 152, 261–264). 2-Deoxyglucose uptake was about 95% depressed in insulin-treated, but not in ‘basal’, cells preincubated with epinephrine plus adenosine deaminase for 60 min in broad agreement with Green's report. However, this depression was caused by a decrease in sugar phosphorylation rather than transport. In similarly incubated cells, transport of 3-O- methylglucose , a sugar analogue not phosphorylated in the adipocytes, was not affected by catecholamine plus adenosine deaminase. However, a decrease in transport of about 60% was observed both in the absence and the presence of insulin when the albumin concentration was high enough and the cell concentration low enough to prevent accumulation of free fatty acids in the medium. In addition, the insulin sensitivity with regard to hexose transport was markedly reduced. Transport was approximately doubled in cells incubated with 5 μM epinephrine in the absence of adenosine deaminase. Thus, epineprine at a high concentration stimulates hexose transport in the absence of adenosine deaminase (presence of adenosine) whereas it inhibits both basal and insulin-stimulated transport in the presence of adenosine deaminase (absence of adenosine).

Insulin binding, glucose oxidation, and methylglucose transport in isolated adipocytes from pregnant rats near term.

To elucidate the mechanism of insulin resistance during late pregnancy, we studied [125I]iodoinsulin binding, [1-14C]glucose oxidation, and 3-O-[methyl-14C]glucose transport in adipocytes isolated from pregnant rats on day 19 or 20 of gestation. Neither the affinity or number of insulin receptors on pregnant rat adipocytes differed from those on age-matched nonpregnant female rats. Insulin-stimulated glucose oxidation was reduced in the pregnant rat adipocytes. The maximum velocity of insulin-stimulated methylglucose transport was also significantly reduced in the pregnant rat adipocytes. These results suggest that insulin resistance in isolated adipocytes from pregnant rats near term is caused by some postreceptor changes, one of which is a reduction in the number and/or mobility of insulin-stimulated hexose transporters.

Glucose 6-phosphate effects on deoxyglucose, glucose and methylglucose transport in rat adipocytes. Evidence for intracellular regulation of sugar transport by glucose metabolites

This study was undertaken to determine whether glucose 6-phosphate can modulate hexose transport in rat adipocytes. The data demonstrated that glucose 6-phosphate decreased hexose transport rates in adipocytes and in adipose plasma membrane vesicles. This effect was not via competitive inhibition. The data provide direct evidence that hexose transport can be inhibited noncompetitively by glucose 6-phosphate.