Differential effect of insulin and elevated glucose level on adenosine transport in rat B lymphocytes
Monika Sakowicz1,
Andrzej Szutowicz2 and
Tadeusz Pawelczyk1
1 Department of Molecular Medicine and 2 Department of Laboratory Medicine, Medical University of Gdansk, 80-211 Gdansk, Poland
Correspondence to: T. Pawelczyk; E-mail: tkpaw{at}amg.gda.pl
 |
Abstract
|
---|
Impaired lymphocyte function is a common feature of human diabetes. Nucleoside transport across the plasma membrane is an essential step during lymphocyte growth and activation. In our study, we evaluated the impact of diabetic conditions on nucleoside transport system in B lymphocytes. Examination of the nucleoside transporters expression level in B lymphocytes isolated from diabetic rats revealed significant changes in their mRNA levels. Experiments performed on B cells cultured in medium containing defined concentrations of glucose and insulin showed that the rENT1 mRNA level was sensitive to extracellular glucose concentration and was not affected by insulin. Increase of glucose concentration from 5 to 20 mM caused a decrease of rENT1 mRNA by 80% and was associated with decreased adenosine uptake by the B cells. The effect of glucose was blocked by PD98059, a MAPK kinase inhibitor. The mRNA levels of rENT2 and rCNT2 were highly dependent on insulin but not on glucose concentration. Exposure of lymphocytes to 10 nM insulin resulted in a 2-fold increase in rENT2 mRNA and a 50% decrease in the rCNT2 mRNA level. Alterations in mRNA levels of rENT2 and rCNT2 were associated with changes in adenosine transport. Insulin-induced changes in expression level of rENT2 were blocked by wortmannin, an inhibitor of phosphatidylinositide 3-kinase, whereas the effect of insulin on rCNT2 was inhibited by PD98059 and to a lesser extend by wortmannin. In summary, impaired nucleoside transport in diabetic B lymphocytes results from alterations in the expression of nucleoside transporters, which are independently and differentially regulated by glucose and insulin.
Keywords: MAP kinase, phosphatidylinositide 3-kinase, rCNT2, rENT1, rENT2
 |
Introduction
|
---|
Adenosine is an endogenous compound capable of altering the function of immune cells (1,2). Numerous experimental data indicate that adenosine can affect T-lymphocyte activation, proliferation, IL-2 production and lymphocyte-mediated cytolysis (36). One of the well-known effects of adenosine is the selective regulation of pro- or anti-inflammatory cytokine release and free radical production (79). Impaired lymphocyte function and enhanced susceptibility to infections is a common feature of human diabetes (10,11). The reason for increased susceptibility of diabetic patients to persistent infections is not fully understood. Some of the altered functions of diabetic lymphocytes can be restored by administration of insulin (12,13). Other changes such as reduced production of IL-2, IL-6 and IL-10 were demonstrated to be induced by raised concentration of glucose (14). Previously, we have shown that metabolism of adenosine and expression level of nucleoside transporters in some tissues of diabetic rat is altered (15,16). These data may suggest that the cellular transport of adenosine might be affected by insulin and/or glucose. Studies on endothelial cells isolated from human diabetic umbilical vein indicated that adenosine transport was reduced by increased glucose concentration, whereas in smooth muscle cells isolated from diabetic umbilical artery, adenosine transport was significantly elevated (17,18). Unfortunately, our knowledge of status of nucleoside transporters in cells of the immune system under diabetic conditions is very limited. Adenosine and other nucleosides are transported across the plasma membrane by specific transport proteins, which can be divided into two categories based on the transport mechanism. Nucleoside transport by equilibrative transporters (ENT) is bidirectional and is driven by the concentration gradient, whereas the function of concentrative transporters (CNT) is based on electrochemical ion gradient, so the nucleoside uptake is coupled to that of sodium ions (19). Based on sensitivity to inhibition by NBTI, the equilibrative transport system is subdivided into two types: the sensitive es and insensitive ei ones. The expression level of particular nucleoside transporters varies depending on the cell type and physiological state (20).
In this report, we describe the changes occurring in expression levels of nucleoside transporters in B lymphocytes isolated from diabetic rats. In order to assess the role of insulin and glucose on adenosine transport, we performed studies on B cells cultured at defined insulin and glucose concentrations. Our findings indicate that insulin and glucose differentially and independently regulate the expression levels of nucleoside transporters and adenosine transport in rat B lymphocytes.
 |
Methods
|
---|
Reagents
Histopaque-1077, insulin, thiobutabarbital sodium (Inactin), penicillin, streptomycin, rapamycin, PD98059, wortmannin, glucose, inulin, nitrobenzyltioinosine, adenosine, inosine, thymidine, cytidine erythro-9-(2-hydroxy-3-nonyl)adenine (an adenosine deaminase inhibitor), RPMI-1640 medium and streptozotocin were obtained from Sigma-Aldrich (Poznan, Poland). [3H]Adenosine was from Amersham (Buckinghamshire, England). Oligo(dT) and dNTP were from Roche Diagnostics (Mannheim, Germany). All primers used were from Integrated Technologies, Inc. (Coralville, IA). Total RNA Prep Plus Kit was from A&A Biotechnology (Gdansk, Poland). Tth DNA polymerase, Tfl DNA polymerase and RNasin were from Promega (Madison, WI). Glucose Hexokinase Reagent Set was from Pointe Scientific, Inc. (Lincoln Park, MI). Fluorescein conjugate mouse anti rat CD2[LFA-2] (clone OX-34) was from Chemicon International (Hofheim, Germany). Fluorescein conjugate Armenian hamster anti-rat CD40 (clone HM40-3), and mouse anti-rat CD19 (clone 1D3) were from BD Biosciences (Heidelberg, Germany).
Animals
Male Wistar rats (200240 g) fed on Altromin C 1000 diet (Altromin, Lage, Germany) were used for all experiments. All animals had free access to food and water.
Experimental diabetes
Diabetes was induced by a single intravenous injection of 75 mg/kg body weight streptozotocin (STZ). STZ was dissolved in 10 mM citrate buffer, pH 4.5. Control rats (hereafter referred to as normal rats) were injected with citrate instead of STZ. On days 1, 5 and 10 after STZ injection, blood glucose levels were measured from tail blood. Only rats with a glucose level of 2030 mM were used for subsequent experiments. On day 10, rats were anesthetized with pentobarbital (40 mg/kg of body weight), the spleen was removed and the splenocytes were isolated.
Cells and culture conditions
Single cell suspension of splenocytes was prepared by pressing spleens through sterilized 20 µm pore size nylon mesh gauze in the presence of sterile saline. Mononuclear cells were isolated by centrifugation of the cell suspension through Histopaque-1077 at 700 g for 30 min at room temperature. Cells found at the saline/Histopaque interface were washed and suspended in RPMI-1640 medium supplemented with 3% BSA. The cells were then separated into adhesive and nonadhesive by the panning method (21), relying on incubation (1 h at 37°C) of cell suspension in the presence of 3% BSA in plastic bottles with surface for adhesive cells (Sarstedt AG & Co., Numbrecht, Germany). Following incubation, the nonadhesive cells were washed out. The adhesive cells were detached by flushing the flask with cold (57°C) phosphate-buffered saline (PBS) and collected by centrifugation (700 g for 10 min) and suspended in RPMI-1640 medium supplemented with penicillin (100 units/ml), streptomycin (100 µg/ml), and 10% fetal bovine serum. The purity of isolated cell fractions was examined by flow cytometry. The adherent fraction (B cells) contained 9597% CD2 (OX-34) negative cells, 8993% CD40 (HM40-3) and 8590% CD19 (1D3) positive cells. The number of viable cells was determined by Trypan Blue dye exclusion. Only cell preparations with a 95% viability or greater were used. Cells were cultured in flat-bottomed culture bottles in a humidified atmosphere containing 5% CO2 at 37°C, at a density of 0.51 x 106 cells/ml in a total volume of 6 ml of RPMI-1640 medium supplemented with antibiotics (at concentrations as stated above) and 10% fetal bovine serum, and containing glucose and insulin at a concentration and time detailed in the figure legends. The compounds examined (insulin and inhibitors) were added to lymphocyte culture in the order, concentration and time detailed in the figure legends. Insulin was dissolved in saline, and inhibitors were dissolved in a small volume (<0.2% of the total volume of culture medium) of DMSO.
Transport measurements
Cells were harvested by centrifugation (700 g for 10 min), washed twice in 15 ml of the appropriate transport buffer containing 20 mM HEPESTris pH 7.4, 130 mM NaCl or choline chloride, 3 mM K2HPO4, 2 mM MgCl2, 1 mM CaCl2, and suspended to a final density of 70 x 105 cells/ml. After suspension in the transport buffer, cells were incubated for 30 min at 24°C. The nucleoside transport was determined by the oil stop procedure (22). The uptake process was initiated by mixing 200 µl of the cell suspension with 10 µl of the 3H-labeled adenosine (12 µCi/nmol). Examination of the time course of adenosine transport in rat T lymphocytes revealed that both Na+-dependent and Na+-independent adenosine uptake was linear, at least throughout the 40 s incubation (not shown); therefore in our transport experiments a 30 s time point was routinely used. The adenosine uptake was terminated by transferring an aliquot of the transport mixture on top of 0.2 ml of oil (silicone fluid with a final density of 1.032 g/ml) in a 0.4 ml microcentrifuge tube (0.4 x 4.5 cm), and immediately centrifuged (5000 g for 1 min) on Beckman MicrofugeTM 11. The tip of the tube containing the cell pellet was cut off and placed into the scintillation vial containing 5 ml of the Sigma-Fluor Universal LSC cocktail (Sigma-Aldrich, Poznan, Poland), and radioactivity was counted. In the transport mixture, the [14C]-labeled inulin (0.25 µCi/ml) was included to correct for the extracellular medium trapped in the pellet (usually 46% of inulin was found in the pellet).
The Na+-dependent adenosine transport was calculated by subtracting those rates measured in the Na+-free buffer (NaCl replaced with equimolar choline chloride) from those measured in the Na+ buffer. Equilibrative NBTI-sensitive (es) adenosine transport was calculated by subtracting those rates measured in choline buffer and the presence of 1 µM NBTI from those measured in the choline buffer and the absence of NBTI. Equilibrative NBTI-insensitive (ei) adenosine transport was measured in the choline buffer and the presence of 1 µM NBTI.
RNase protection assay
Changes in the mRNA level of each nucleoside transporter were analyzed by ribonuclease protection technique using Multi Nuclease Protection Assay (Multi-NPA, Ambion) with ß-actin as a reference template. Probes for nucleoside transporters and ß-actin used in RNase protection assay were prepared by PCR as described previously (16). Amplified DNA fragments were 406, 404, 399, 390 and 511 bp for rENT1, rENT2, rCNT1, rCNT2 and ß-actin, respectively. Usually 1015 µg of total RNA was hybridized to the appropriate nucleoside transporter and ß-actin probes according to the manufacturer's protocol. Protected RNA fragments were fractionated by electrophoresis on 8 M urea/6% polyacrylamide gel and transferred to a positively charged nylon membrane. The hybridized probes were immunodetected, visualized and analyzed using the Gel Doc 2000 system (Bio-Rad, Hercules, CA) and the computer program Quantity One (Bio-Rad). The relative expression level of a given nucleoside transporter (NT) gene was presented as a ratio of NT/ß-actin probe.
Analytical
Protein concentrations were determined by the method of Bradford (23) with bovine serum albumin as a standard. The DNA and RNA concentrations were determined by measuring the absorbance at 260 nm. Glucose was measured with the hexokinase method using the Pointe Scientific Kit.
Statistical analysis
The statistical analysis was carried out using the STATISTICA 5PL statistical package (StatSoft). Statistical significance was determined using the t-test. P-values < 0.05 were considered as significant.
 |
Results
|
---|
Expression level of nucleoside transporters in diabetic B lymphocytes isolated from diabetic rats
In order to examine the expression level of nucleoside transporters (NT), we isolated total RNA from B lymphocytes prepared from spleens of normal and streptozotocin (STZ)-induced diabetic rats. RNase protection assays revealed that on day 10 after STZ administration, mRNA levels of rENT1 and rENT2 in diabetic B cells were lowered by 80 and 50%, respectively (Fig. 1). The level of rCNT2 mRNA was increased 2.1-fold in diabetic B cells. In our assay we did not observe any signal from the probe to rCNT1. This may indicate that the mRNA for rCNT1 transporter was absent in B lymphocytes or that its level was very low (not shown). Changes in NT mRNA levels observed in diabetic cells would indicate that alterations in glucose and/or insulin level may be the factors responsible for observed alterations in expression of NT. However, direct action of STZ on NT genes should also be considered. It was reported that lymphocytes from STZ-induced diabetic mice or cells incubated in vitro with STZ contained numerous chromosomal abnormalities indicative of DNA strand breaks (24). In order to avoid a possible direct effect of STZ, and to discriminate the effect of glucose and insulin on expression level of NT, subsequent experiments were performed on B lymphocytes cultured in medium containing defined concentrations of glucose and insulin.

View larger version (25K):
[in this window]
[in a new window]
|
Fig. 1. The mRNA level of nucleoside transporters in B lymphocytes isolated from spleens of normal and STZ-induced diabetic rats. (A) The presented RNase protection assays are representative of those obtained in experiments performed on RNA isolated from lymphocytes of normal (N) and diabetic (D) rats. The positions of ß-actin and nucleoside transporter (NT) bands are indicated. (B) The quantified results of RNase protection assays normalized to ß-actin mRNA levels. The data represent the mean ± SD from three independent experiments. *P < 0.02, D versus N.
|
|
The effect of glucose on expression level of nucleoside transporters and adenosine transport in cultured rat B lymphocytes
B lymphocytes isolated from spleens of normal rats cultured for 4 days at increased concentrations of glucose and in the presence of 10 nM insulin showed a reduced level of rENT1 mRNA. Alterations in rENT1 mRNA level were associated with decreased es adenosine transport in B cells (Fig. 2). The effect of high glucose on rENT1 expression level was reversible, and changing the culture medium to low glucose (5 mM) medium resulted in restoration of the rENT1 mRNA level seen at 5 mM glucose. Maximal effect of 20 mM glucose on rENT1 mRNA level was visible on the third day (Fig. 3). The effect of glucose was not dependent on insulin, and the same glucose-induced changes in rENT1 mRNA level were observed in the presence or absence of 10 nM insulin (not shown). Changes in concentration of glucose neither affected the mRNA levels of rENT2 and rCNT2 nor altered the Na+-dependent and NBTI-insensitive equilibrative adenosine transport (data not shown).

View larger version (32K):
[in this window]
[in a new window]
|
Fig. 2. Dose-dependent course of glucose action on the rENT1 mRNA level in cultured rat B lymphocytes. Cells were cultured as described in the Methods for 3 days in the presence of glucose at the concentrations indicated. On day 4, cells were harvested, total RNA was extracted and RNase protection assay was performed. (A) Representative RNase protection assay; positions of ß-actin and rENT1 bands are indicated. (B) The quantified results of RNase protection assays normalized to ß-actin mRNA. The data represent the mean ± SD from four independent experiments. *P < 0.05 for 7 mM glucose versus 5 mM glucose; **P < 0.001 for 10 mM glucose versus 7 mM glucose; ***P < 0.02 for 15 mM glucose versus 10 mM glucose; ****P < 0.05 for 20 mM glucose versus 15 mM glucose.
|
|

View larger version (21K):
[in this window]
[in a new window]
|
Fig. 3. Time course of glucose action on the abundance of rENT1 mRNA in cultured rat B lymphocytes and adenosine es transport. Cells were cultured for 2 days in the presence of 5 mM glucose. On day 3 (time 0), cells were harvested and transferred to the culture medium containing 20 mM glucose, and incubated for 3 days. After 96 h of incubation, cells cultured at 20 mM glucose were harvested and transferred to the medium containing 5 mM glucose. At the indicated time-points, 2 x 106 cells were withdrawn from the cell culture. Half of the cells were used for measurements of adenosine (50 µM) uptake (A). From the other half, RNA was extracted and RNase protection assay was performed (B). The data represent the mean ± SD from three independent experiments. (A) *P < 0.0003 for 24 h versus time 0; **P < 0.004 for 48 h versus 24 h; #P < 0.003 for 120 h versus 96 h; ##P < 0.0003 for 144 h versus 120 h. (B) *P < 0.001 for 24 h versus time 0; **P < 0.05 for 72 h versus 48 h; #P < 0.006 for 120 h versus 96 h; ##P < 0.03 for 144 h versus 120 h.
|
|
The effect of insulin on expression level of nucleoside transporters and adenosine transport in cultured rat B lymphocytes
In order to examine the effect of insulin on NT expression in B lymphocytes, the cells were cultured at 5 mM glucose and various concentrations of insulin. Our experiments showed that the mRNA levels of rENT2 and rCNT2 were highly dependent on insulin (Fig. 4). The same effect of insulin on NT mRNA level was observed in cells cultured at 520 mM concentration of glucose (not shown). The character of insulin-induced changes in the expression of rENT2 and rCNT2 differed significantly. Exposition of B lymphocytes to 10 nM insulin resulted in a 2-fold increase in rENT2 mRNA and a 50% decrease in the rCNT2 mRNA level (Fig. 4). Examination of the insulin dose response effect on NT expression revealed that a maximal effect could be observed at 10 nM insulin, and only a slight increase was seen with 100 nM insulin (Fig. 5A). The maximal effect of 10 nM insulin on mRNA levels of rENT2 and rCNT2 was observed at the 7th and 5th hour, respectively (Fig. 5B). Alterations in mRNA levels of rENT2 and rCNT2 were associated with changes in adenosine transport. Cells cultured in the presence of insulin showed increased equilibrative NBTI-insensitive (ei) adenosine uptake and decreased Na+-dependent transport (Fig. 6). The nature of Na+-dependent adenosine transport was assessed based on results from experiments with competing nucleosides and NBTI. The Na+-dependent adenosine uptake was inhibited by 26% in the presence of 1 µM NBTI (Fig. 6C). Pyrimidine nucleosides (thymidine and cytidine) at a concentration of 200 µM competitively inhibited Na+-dependent adenosine (10 µM) uptake by only 20% (Fig. 6D). These data indicate that in B lymphocytes the Na+-dependent adenosine transport has mostly cif nature, but the presence of cib transporting system broad selectivity for purine and pyrimidine nucleosides cannot be excluded.

View larger version (20K):
[in this window]
[in a new window]
|
Fig. 4. Effect of insulin on ENT2 and CNT2 mRNA level in cultured rat B lymphocytes. B cells were cultured for 3 days in the presence of 5 mM glucose and the absence (Ins) or presence (+Ins) of 10 nM insulin. (A) Representative RNase protection assays: the positions of ß-actin and nucleoside transporter (NT) bands are indicated. (B) The quantified results of RNase protection assays normalized to ß-actin mRNA. The data represent the mean ± SD from four independent experiments. *P < 0.003 (+Ins) versus (Ins).
|
|

View larger version (21K):
[in this window]
[in a new window]
|
Fig. 5. Dose- and time-dependent courses of insulin action on ENT2 and CNT2 mRNA levels in cultured rat B lymphocytes. (A) Cells were cultured for 2 days in the presence of 5 mM glucose and the absence of insulin. Insulin at the indicated concentrations was added to the culture medium on day 3, and cells were cultured for another 10 h, harvested and NT mRNA was determined. Concentration of insulin in RPMI-1640 medium supplemented with 10% FBS varied in the range of 49 x 1011 M. Insulin in this concentration had no measurable effect on NT mRNA level (not shown). (B) Cells were cultured for 2 days in the presence of 5 mM glucose and 10 mM insulin was added to the culture medium on day 3 (time 0). Cells were harvested at the times indicated and NT mRNAs were quantified by RNase protection assay. The data represent the mean ± SD from three independent experiments. (A) *P < 0.003 for ENT2 mRNA level at 5 x 109 M insulin versus ENT2 mRNA level at 109 M insulin; **P < 0.006 for ENT2 mRNA level at 108 M insulin versus ENT2 mRNA level at 5 x 109 M insulin; #P < 0.04 for CNT2 mRNA level at 5 x 109 M insulin versus CNT2 mRNA level at 109 M insulin; ##P < 0.03 for CNT2 mRNA level at 108 M insulin versus CNT2 mRNA level at 5 x 109 M insulin. (B) *P < 0.008 for 3 h versus time 0; **P < 0.007 for 5 h versus 3 h; ***P < 0.001 for 7 h versus 5 h; #P < 0.001 for 2 h versus time 0; ##P < 0.003 for 4 h versus 2 h; ###P < 0.04 for 5 h versus 4 h.
|
|

View larger version (21K):
[in this window]
[in a new window]
|
Fig. 6. Na+-dependent and equilibrative ei adenosine transport in cultured B lymphocytes as a function of insulin, NBTI and thymidine presence. Na+-dependent (A) and equilibrative ei (B) adenosine transport in cells cultured for 2 days at 5 mM glucose and the presence (+) or absence () of 10 nM insulin. The effect of 1 µM NBTI (C) and 200 µM thymidine (D) on Na+-dependent adenosine (10 µM) uptake by cells cultured in the presence of 10 nM insulin. The data represent the mean ± SD from at least five independent experiments. *P < 0.004 for (+Ins) versus (Ins); **P < 0.005 for (+NBTI) versus (NBTI); ***P < 0.03 for (+Thymidine) versus (Thymidine).
|
|
Effect of insulin and glucose on the kinetics of adenosine transport
In order to assess the impact of variation in concentration of glucose and insulin on nucleoside transport in B cells, adenosine transport was measured at nucleoside concentrations ranging from 1 to 300 µM in cells cultured in the presence of various concentrations of glucose and insulin. The concentration-dependent uptake of adenosine was saturable and conformed to MichaelisMenten kinetics. Kinetic parameters calculated by non-linear regression of the v versus v/s plots indicate that alterations in the glucose and insulin level induced changes in the Vmax but not in the Km value (Table I). This suggested that insulin and glucose influenced the number of nucleoside transporters in the cell but not the affinity for adenosine. This assumption is consistent with the observation that both insulin and glucose differentially and independently affected the NT mRNA levels in B cells (Figs 2 and 4). Analysis of the adenosine transport in cells cultured at high and low glucose and in the absence or presence of insulin indicated that insulin affects the Na+-dependent transporter(s) and NBTI-insensitive ei transporter (Table I). On the other hand, the NBTI-sensitive es transporter appeared to be sensitive to changes in glucose level.
Insulin and glucose signaling
The phosphatidylinositide 3-kinase (PI3K) pathway and the MAPK pathway are the main routes downstream of the insulin receptor. To define the key steps that are used by insulin to regulate expression of rENT2 and rCNT2, we used specific inhibitors of distinct steps of insulin signaling pathways. Prior treatment of the cells with 0.1 µM wortmannin (an inhibitor of PI3K) before the addition of insulin completely blocked insulin-induced elevation of rENT2 mRNA level and an increase of ei adenosine uptake (Fig. 7A). Rapamycin (an inhibitor of mTOR) and PD98059 (an inhibitor of MEK) did not affect the insulin-induced changes in rENT2 mRNA level and ei adenosine uptake. Insulin-induced changes in rCNT2 mRNA level and in Na+-dependent adenosine transport were blocked by pretreatment of the cells with 10 µM PD98059 and to a lesser extend by 0.1 µM wortmannin (Fig. 7B). These findings suggest that the MAPK/PI3K pathway plays a significant role in the regulation of rCNT2 by insulin. To address the question of whether glucose utilized the PI3K/p70S6K/MAPK pathway to regulate the rENT1, we incubated the cells with 20 mM glucose and the presence of employed inhibitors which were added to the culture medium 30 min before raising the glucose concentration. The results presented in Fig. 8 indicate that the effect of 25 mM glucose on rENT1 mRNA level and es adenosine transport was blocked by PD98059, whereas 0.1 µM rapamycin had no effect on glucose-induced changes. Only slight inhibition of the effect of glucose was observed on addition of 0.1 µM wortmannin.

View larger version (27K):
[in this window]
[in a new window]
|
Fig. 7. The effect of wortmannin, PD98059 and rapamycin on insulin-induced rENT2 and rCNT2 mRNA levels, and equilibrative ei and Na+-dependent adenosine transports in cultured B lymphocytes. The compounds examined were added to B cell culture 30 min before the addition of 10 nM insulin. The final concentrations of wortmannin, PD98059 and rapamycin were 0.1, 10 and 0.1 µM, respectively. Control cells were treated with an appropriate volume of solvent (DMSO) used for solubilizing the inhibitors. After addition of insulin, the cells were cultured for 8 h, then adenosine (50 µM) uptake was measured after harvesting, and rENT2 (A) and rCNT2 (B) mRNA levels were determined. Representative RNase protection assays for each transporter mRNA are presented and the positions of ß-actin and nucleoside transporter bands are indicated. The data represent the mean ± SD from at least four independent experiments. (A) *P < 0.02 relative to ENT2 mRNA level and Ado uptake in cells cultured in the presence of insulin and the absence of wartmannin. (B) *P < 0.04 relative to CNT2 mRNA level and Ado uptake in cells cultured in the presence of insulin alone; **P < 0.004 relative to CNT2 mRNA level and Ado uptake in cells cultured in the presence of insulin alone.
|
|

View larger version (31K):
[in this window]
[in a new window]
|
Fig. 8. Effect of wortmannin, PD98059 and rapamycin on glucose-induced rENT1 mRNA level and equilibrative es adenosine transport in cultured B lymphocytes. B cells were cultured for 2 days at 5 mM glucose and the presence of 10 nM insulin. The compounds examined were added to cell culture 30 min before the addition of glucose to reach the 25 mM concentration. The final concentrations of wortmannin, PD98059 and rapamycin were 0.1, 10 and 0.1 µM, respectively. Control cells were treated with an appropriate volume of solvent (DMSO) used for solubilizing the inhibitors. Cells were cultured for 24 h at 25 mM glucose, and after harvesting, adenosine (50 µM) uptake was measured and rENT1 mRNA level was determined. A representative RNase protection assay is presented at the top. The data represent the mean ± SD from at least four independent experiments. *P < 0.0002 relative to ENT1 mRNA level and Ado uptake in cells cultured in the presence of 25 mM glucose alone; **P < 0.04 relative to ENT1 mRNA level and Ado uptake in cells cultured in the presence of 25 mM glucose alone.
|
|
 |
Discussion
|
---|
Most of the known immunomodulatory effects of adenosine are thought to be mediated through binding to specific surface receptors. However, some actions of adenosine seem to require its uptake by the cell. Studies on mice deficient for the A2a receptor subtype showed that in peritoneal macrophages, adenosine inhibits IL-12 and TNF-
production via receptor-dependent and independent mechanisms (8). The requirement of adenosine uptake for induction of several intracellular processes in other cell types was also reported, including apoptosis of mouse neuroblastoma cells (25), growth arrest of rat pituitary cells (26) and, depending on sympathetic neurons state, both induction and prevention of apoptosis (27). On the other hand, nucleoside transporters, by removing adenosine from the extracellular space, may terminate its action on cell surface receptors. Conversely, if the supply/demand ratio of energy falls and adenosine accumulates in the cell, the equilibrative transporters may release adenosine to the extracellular matrix. Therefore, factors that regulate the nucleoside transporters both at the transcriptional and protein levels may alter both the receptor-dependent and independent action of adenosine, while expression of the particular nucleoside transporter varies depending on cell type and physiological state. Studies performed on B cell lines and murine bone macrophages showed that these cells express both the concentrative (Na+-dependent) and equilibrative (Na+-independent) nucleoside transport systems (28). The presence of mRNA for ENT1, ENT2 and CNT2 was detected in several human hematological cell lines and normal leukocytes (29). In this study, the presence of rENT1, rENT2 and rCNT2, but not rCNT1, mRNA in rat B lymphocytes is reported.
The rate of endogenous nucleotide synthesis in hematopoietic cells is low; therefore, for proper functioning they require nucleoside uptake to meet their metabolic demands (30,31). Proliferation and differentiation of lymphocytes are associated with DNA and RNA synthesis, which require exogenous nucleosides. It was reported that macrophage proliferation and activation require selective regulation of nucleoside transport systems (28). Signals that induced proliferation led to up-regulation of the equilibrative transporters, whereas activation of macrophages was associated with down-regulation of equilibrative transporters (es) and up-regulation of concentrative ones (32,33). Moreover, the essentialness of es nucleoside transport was documented in experiments demonstrating that M-CSF-induced proliferation of macrophages was blocked by NBTI treatment (32). In the current study, we present evidence that an elevated level of glucose suppresses expression of rENT1 transporter in B lymphocytes, leading to significant impairment of adenosine uptake. The inhibitory effect of elevated glucose levels on adenosine transport and expression of rENT1 was blocked by PD98059 (an inhibitor of MEK-1), indicating that the MAP kinase pathway is involved in the regulation of rENT1. The ability of elevated glucose to induce MAP kinases is consistent with previous reports on human umbilical vein endothelial cells (34), rat aorta smooth muscle cells (35) and glomerular mesangial cells (36). The study of Montecinos et al. (36) showed that elevated glucose increased activity of PKC and the phosphorylation level of ERK1 and ERK2 in human fetal endothelial cells. On the other hand, it has been demonstrated that activation of PKC
and
led to phosphorylation of MAP kinases in rat mesangial cells (38). However, the exact mechanism that glucose utilizes to affect gene expression remains obscure. It has been reported that glucose 6-phosphate itself is a signaling molecule (37), but the subsequent steps of the glucose signaling pathway are still elusive.
Insulin did not affect the expression of rENT1 nor the es adenosine transport system. On the other hand, insulin down-regulated the expression level of rCNT2 transporter and up-regulated the level of rENT2. Our results indicate that the effect of insulin on expression level of rENT2 and ei adenosine transport is blocked by wortmannin, an inhibitor of phosphatidylinositide 3-kinase (PI3K), but not by an inhibitor (rapamycin) of mTOR. This indicates that transmission of the insulin signal to rENT2 does not involve another element downstream of the PI3K pathway, namely p70 ribosomal S6 kinase (p70S6K). Rapamycin, by inactivating mTOR, potently inhibits phosphorylation and activation of p70S6K (38). Insulin-induced suppression of the rCNT2 expression was totally blocked by PD98059, indicating that insulin controls rCNT2 expression levels by signaling through MAPK pathway. However, the small inhibition of insulin effect visible in the presence of wortmannin indicates that the PI3K-dependent pathway is also involved in regulation of rCNT2 by insulin. Further work is required to delineate the role of particular elements of these two signaling pathways in transmitting the insulin signal to rCNT2 in rat B lymphocytes. Despite the significant effects of insulin on expression of rENT2 and rCNT2, insulin did not significantly affect total adenosine uptake by B cells because the ei transport system and concentrative cif transport were altered to a similar extend (Table I). Conversely, it might be assumed that B cell adenosine outflow is sensitive to the action of insulin because nucleosides can leave cells only by equilibrative transport systems. Under in vitro conditions, the capability of B lymphocytes to produce and release adenosine was demonstrated (39). Obviously the nucleoside fate in the cell beside transport processes highly depends on its metabolism, especially phosphorylation. However, in the case of phosphorylated forms of nucleosides, the multidrug resistance-associated proteins (MRP) are most likely responsible for releasing them from the cell (40).
The differential effects of glucose and insulin on expression level of nucleoside transporters and adenosine transport observed in our experiments indicate that nucleoside transport in B lymphocytes is significantly impaired in diabetes. Hyperglycemia and hypoinsulinemia may lead in B cells to significant reduction both the adenosine uptake and outflow, possibly resulting in impairment of autocrine action of adenosine. Highly reduced thymidine uptake associated with impaired lymphocyte proliferation was observed in peripheral blood mononuclear cells from patients with non-insulin dependent diabetes mellitus (41). Moreover, hyporesponsiveness of mononuclear cells to stimulation was reported in both insulin-dependent and independent diabetes (42,43). Abnormalities in the function of immune cells have been considered to be a major cause of increased risk for infections in diabetic patients (44), but the information on the particular alterations is incomplete and contradictory. Altered cellular and humoral immunity was observed in diabetic patients and in animal experimental diabetes, while general impairment in total IgM and IgG production in response to coxasackievirus B4 was observed in mouse with the genetic predisposition to develop diabetes mellitus (45). However, in pediatric patients with type 1 diabetes no differences in IgG- and IgM-antibody classes for coxasackievirus were detected comparing to their matched health control group (46). Recently reported results from a controlled vaccination study demonstrated a significant impairment of the humoral immune response to T-cell-dependent antigens in type-1 but not type-2 diabetic patients (47). The study demonstrated that reduced antibody production was associated with impaired ability of diabetes type-1 T cells to produce both INF-
and IL-13 in response to stimulation with the vaccination antigen. Other reports described impaired cellular immune response in patients with type 2 diabetes. It was reported that patients with type 2 diabetes displayed a significant reduction in percentage of activated cells possessing receptor for IL-2 and down-regulation of basal cytokine (IL-6, TNF-
) production in blood cells (48,49).
In summary, our study demonstrates that the expression level of adenosine transporters in B lymphocytes is independently and differentially regulated by insulin and glucose. Under diabetic-like conditions, i.e. hyperglycemia and the absence of insulin, the changes occurring in expression levels of nucleoside transporters in B cells lead to diminished adenosine uptake. Although the relationship between altered nucleoside transport and the diabetic immunodeficiency is highly speculative, we assume that disturbances in B cell nucleoside transport occurring under diabetic conditions might contribute to pathomechanisms underlying impaired humoral immunity. Further studies are necessary in order to assess the impact of altered nucleoside transport on immune cell function.
 |
Acknowledgements
|
---|
The authors thank Filip Golebiowski for his excellent assistance in preparing the manuscript. This work was supported by the State Committee for Scientific Research (KBN) grant No. 3 P05A 055 24.
 |
Abbreviations
|
---|
Ado | adenosine |
ci | concentrative nucleoside transport insensitive to NBTI |
cif | purine-preferring concentrative transport insensitive to NBTI |
CNT | concentrative (Na+-dependent) nucleoside transporter |
ei | equilibrative nucleoside transport system insensitive to inhibition by NBTI |
ENT | equilibrative nucleoside transporter |
es | equilibrative nucleoside transport system sensitive to inhibition by NBTI |
NBTI | nitrobenzylthioinosine |
NT | nucleoside transporter |
STZ | streptozotocin |
 |
Notes
|
---|
Transmitting editor: S.H.E. Kaufmann
Received 21 July 2004,
accepted 9 November 2004.
 |
References
|
---|
- Hershfield, M. S. and Mitchell, B. S. 1995. Immunodeficiency diseases caused by adenosine deaminase deficiency and purine nucleoside phosphorylase deficiency. In Scriver, C. R., Beaudet, A. L., Sly, W. S. and Valle, D. eds, The Metabolic and Molecular Bases of Inheritated Disease, p. 1725. McGraw-Hill Inc., New York.
- Cronstein, B. N. 1994. Adenosine, an endogenous anti-inflamantory agent. J. Appl. Physiol. 76:5.[Abstract/Free Full Text]
- Wolberg, G., Zimmerman, T. P., Hiemstra, K., Winston, M. and Chi, L.-C. 1975. Adenosine inhibition of lymphocyte-mediated cytolysis: possible role of cyclic adenosine monophosphate. Science 187:957.[ISI][Medline]
- Dos Reis, G. A., Nobrega, A. F. and Paes de Carvalho, R. 1986. Purinergic modulation of T-lymphocyte activation: differential susceptibility of distinct activation steps and correlation with intracellular 3'5'-cyclic adenosine monophosphate accumulation. Cell Immunol. 101:213.[CrossRef][ISI][Medline]
- Antonysamy, M. A., Moticka, E. J. and Ramkumar, V. 1995. Adenosine acts as an endogenous modulator of IL-2 dependent proliferation of cytotoxic T lymphocytes. J. Immunol. 155:2813.[Abstract]
- Apasov, S., Koshiba, M., Redegeld, F. and Sitkovsky, M. 1995. Role of extracellular ATP and P1 and P2 classes of purinergic receptors in T-cell development and cytotoxic T lymphocyte effector functions. Immunol. Rev. 146:5.[CrossRef][ISI][Medline]
- Hasko, G., Szabo, C., Nemeth, Z. H., Kvetan, V., Pastores, S. M. and Vizi, E. S. 1996. Adenosine receptor agonist differentially regulate IL-10, TNF-
and nitric oxide production in Raw-264.7 macrophages and in endoxemic mice. J. Immunol. 157:4634.[Abstract]
- Hasko, G., Kuhel, D. G., Chen, J.-F., Schwarzschild, M. A., Deith, E. A., Mabley, J. G., Marton, A. and Szabo, C. 2000. Adenosine inhibits IL-12 and TNF-
production via adenosine A2a receptor-dependent and independent mechanisms. FASEB J. 14:2065.[Abstract/Free Full Text]
- Cain, B. S., Harken, A. H. and Meldrum, D. R. 1999. Therapeutic strategies to reduce TNF-
mediated cardiac contractile depression following ischemia and reperfusion. J. Mol. Cell. Cardiol. 31:931.[CrossRef][ISI][Medline]
- Kraine, M. R. and Tisch, R. M. 1999. The role of environmental factors in insulin dependent diabetes mellitus: an unresolved issue. Enviromen. Health Persp. 107:770.
- Larkin, J. G., Frier, B. M. and Ireland, J. 1985. Diabetes mellitus and infection. Postgrad. Med. J. 61:233.[ISI][Medline]
- Muller, C. et al. 1989. Effects of cyclosporine A upon humoral and cellular immune parameters in insulin-dependent diabetes mellitus type 1: a long-term follow-up study. J. Endocrinol. 121:177.[Abstract]
- Korfel, J., Kinalska, I., Rogowski, F. and Citko, A. 1990. Cellular immunity in insulin-dependent diabetes mellitus. Pol. Tyg. Lek. 45:373.[Medline]
- Reinhold, D., Ansorge, S. and Schlaicher, E. D. 1996. Elevated glucose levels stimulate transforming growth factor-ß1 (TGF-ß1), suppress interleukin IL-2, IL-6 and IL-10 production and DNA synthesis in peripheral blood mononuclear cells. Horm. Metab. Res. 28:267.[ISI][Medline]
- Pawelczyk, T., Sakowicz, M., Szczepanska-Konkel, M. and Angielski, S. 2000. Decreased expression of adenosine kinase in streptozotocin-induced diabetes mellitus rats. Arch. Biochem. Biophys. 375:1.[CrossRef][ISI][Medline]
- Pawelczyk, T., Podgorska, M. and Sakowicz, M. 2003. The effect of insulin on expression level of nucleoside transporters in diabetic rats. Mol. Pharmacol. 63:81.[Abstract/Free Full Text]
- Sobrevia, L., Jarvis, S. M. and Yudilevich, D. L. 1994. Adenosine transport in cultured human umbilical vein endothelial cells is reduced in diabetes. Am. J. Physiol. 267:C39.[ISI][Medline]
- Aguayo, C., Flores, C., Parodi, J., Rojas, R., Mann, G. E., Pearson, J. D. and Sobrevia, L. 2001. Modulation of adenosine transport by insulin in human umbilical artery smooth muscle cells from normal or gestational diabetic pregnancies. J. Physiol. 534:243.[Abstract/Free Full Text]
- Baldwin, S. A., Mackey, J. R., Cass, C. E. and Young, J. D. 1999. Nucleoside transporters: molecular biology and implications for therapeutic development. Mol. Med. Today 5:216.[CrossRef][ISI][Medline]
- Pennycooke, M., Chaudary, N., Shuralyova, I., Zhang, Y. and Coe, I. R. 2001. Differential expression of human nucleoside transporters in normal and tumor tissue. Biochem. Biophys. Res. Commun. 280:951.[CrossRef][ISI][Medline]
- Severson, C. D., Burg, D. L., Lafrenz, D. E. and Feldbush, T. L. 1987. An alternative method of panning for rat B lymphocytes. Immunol. Lett. 15:291.[CrossRef][ISI][Medline]
- McGivan, J. D. 1989. Transport of alanine across hepatocyte plasma membranes. Methods Enzymol. 174:31.[ISI][Medline]
- Bradford, M. M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of proteindye binding. Anal. Biochem. 72:248.[CrossRef][ISI][Medline]
- Gaulton, G. N., Schwarta, J. L. and Eardley. D. D. 1985. Assessment of the diabetogenic drugs alloxan and streptozotocin as models for the study of immune defects in diabetic mice. Diabetologia 28:769.[ISI][Medline]
- Schrier, S. M., van Tilburg, E. W., van der Meulen, H., Ijzerman, A. P., Mulder, G. J. and Nagelkerke. J. F. 2001. Extracellular adenosine-induced apoptosis in mouse neuroblastoma cells: studies on involvement of adenosine receptors and adenosine uptake. Biochem. Pharmacol. 61:417.[CrossRef][ISI][Medline]
- Lewis, M. D., Hepburn, P. J. and Scanlon, M. F. 1997. Epidermal growth factor protects GH3 cells from adenosine induced growth arrest. Mol. Cell. Endocrinol. 127:137.[CrossRef][ISI][Medline]
- Wakade, A. R., Przywara, S. D. and Wakade, T. D. 2001. Intracellular, nonreceptor-mediated signaling by adenosine: induction and prevention of neuronal apoptosis. Mol. Neurobiol. 23:137.[CrossRef][ISI][Medline]
- Pastor-Anglada, M., Casado, F. J., Valdes, R., Mato, J., Garcia-Manteiga, J. and Molina, M. 2001. Complex regulation of nucleoside transporter expression in epithelial and immune system cells. Mol. Memb. Biol. 18:81.[CrossRef][ISI][Medline]
- Molina-Arcas, M., Bellosillo, B., Casado, F. J., Montserrat, E., Gil, J., Colomer, D. and Pastor-Anglada, M. 2003. Fludarabine uptake mechanisms in B-cell chronic lymphocytic leukemia. Blood 101:2328.[Abstract/Free Full Text]
- Fox, I. H. and Kelley, W. N. 1978. The role of adenosine and 2'-deoxyadenosine in mammalian cells. Annu. Rev. Biochem. 47:655.[CrossRef][ISI][Medline]
- Murry, A. W. 1971. The biological significance of purine salvage. Ann. Rev. Biochem. 40:811.[CrossRef][ISI][Medline]
- Soler, C., Garcia-Manteiga, J., Valdes, R., Xaus, J., Comalada, M., Casado, F. J., Pastor-Anglada, M., Celada, A. and Felipe, A. 2001. Macrophages require different nucleoside transport systems for proliferation and activation. FASEB J. 15:1979.[Abstract/Free Full Text]
- Soler, C. et al. 2001. Lipopolysacharide-induced apoptosis of macrophages determines the up-regulation of concentrative nucleoside transporters Cnt1 and Cnt2 through tumor necrosis factor-
-dependent and -independent mechanisms. J. Biol. Chem. 276:30043.[Abstract/Free Full Text]
- Montecinos, P. V., Aguayo, C., Flores, C., Wyatt, A. W., Pearson, J. D., Mann, G. E. and Sobrevia, L. 2000. Regulation of adenosine transport by D-glucose in human fetal endothelial cells: involvement of nitric oxide, protein kinase C and mitogen-activated protein kinase. J. Physiol. 529:777.[Abstract/Free Full Text]
- Natarajan, R., Scott, S., Bai, W., Yerneni, K. K. and Nadler, J. 1999. Angiotensin II signaling in vascular smooth muscle cells under high glucose conditions. Hypertension 33:378.[Abstract/Free Full Text]
- Haneda, M., Araki, S., Togawa, M., Sugimoto, T., Isono, M. and Kikkawa. R. 1997. Mitogen-activated protein kinase cascade is activated in glomeruli of diabetic rats and glomerular mesangial cells cultured under high glucose conditions. Diabetes 46:847.[Abstract]
- Vaulon, S., Vasseur-Cognet, M. and Kahn, A. 2000. Glucose regulation of gene trascription. J. Biol. Chem. 275:31555.[Free Full Text]
- Abraham, R. T. and Wiederrecht, G. J. 1996. Immunopharmacology of rapamycin. Annu. Rev. Immunol. 14:483.[CrossRef][ISI][Medline]
- Barankiewicz, J., Ronlov, G., Jimenez, R. and Gruber, H. 1990. Selective adenosine release from human B but not T lymphoid cell line. J. Biol. Chem. 265:15738.[Abstract/Free Full Text]
- Kruh, G. D. and Belinsky, M. G. 2003. The MRP family of drug efflux pumps. Oncogene 22:7537.[CrossRef][ISI][Medline]
- Chang, F.-Y. and Shaio, M.-F. 1995. Decreased cell-mediated immunity in patients with non-insulin-dependent diabetes mellitus. Diab. Res. Clin. Prac. 28:137.[CrossRef][ISI]
- MacCuish, A. C., Urbaniak, S. J., Cambell, C. J., Duncan, L. P. J. and Irvine, W. J. 1974. Phytohemagglutinin transformation and circulating lymphocyte subpopulations in insulin-dependent diabetics. Diabetes 23:708.[ISI][Medline]
- Casey, J. I., Heeter, B. J. and Klyshevich, K. A. 1977. Impaired response of lymphocytes of diabetic subjects to antigen of Staphylococcus aureus. J. Inf. Dis. 136:495.[ISI][Medline]
- Puxty, J. A. H. and Fox, R. A. 1984. Diabetes and infection. In Fox, R. A. ed., Immunology and Infection in the eldery. p. 79. Churchill Livingstone, London.
- Montgomery, L. B. and Loria, R. M. 1986. Humoral immune response in hereditary and overt diabetes mellitus. J. Med. Virol. 19:255.[ISI][Medline]
- Hyoty, H., Huupponen, T., Kotola, L. and Leinikki, P. 1986. Humoral immunity against viral antigens in type 1 diabetes: altered IgA-class immune response against Coxsackie B4 virus. Acta Pathol. Microbiol. Immunol. Scand. 94:83.[ISI]
- Eibl, M. M., Spatz, M., Fischer, G. F., Mayr, W. R., Samstag, A., Wolf, H. M., Schernthaner, G. and Eibl, M. M. 2002. Impaired primary immune response in type-1 diabetes: results from a controlled vaccination study. Clin. Immunol. 103:249.[CrossRef][ISI][Medline]
- Pozzilli, P., Gale, E. A., Visalli, N., Baroni, M., Crovari, P., Frighi, V., Cavallo, M. G. and Andreani, D. 1986. The immune response to influenza vaccination in diabetic patients. Diabetologia 29:850.[CrossRef][ISI][Medline]
- Pickup, J. C., Chusney, G. D., Thomas, S. M. and Burt, D. 2000. Plasma interleukin-6, tumor necrosis factor alfa and blood cytokine production in type 2 diabetes. Life Sci. 67:291.[CrossRef][ISI][Medline]