Expression of selected glucose transporters in peripheral blood lymphocytes in patients with type 2...

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ORIGINALAgnieszka Sobczyk-Kopcioł1, Leszek Szablewski1, Bożenna Oleszczak1,Barbara Grytner-Zięcina1, Beata Mrozikiewicz-Rakowska2,Waldemar Karnafel2, Ewa Warnawin3

1Department of General Biology and Parasitology, Medical University of Warsaw, Poland, 2Chair and Clinic of Gastroenterologyand Metabolic Disorders, Medical University of Warsaw, Poland, 3Department of Pathophysiology and Immunology,Rheumatology Institute in Warsaw, Poland

Expression of selected glucose transporters inperipheral blood lymphocytes in patients withtype 2 diabetes mellitus managed with insulin

AbstractBackground. Glucose is transported from blood into cellsby means of transmembrane transporting proteins whichplay an important role in the maintenance of bodyhomoeostasis. The aim of the study was to evaluate thepresence and contents of selected isoforms of glucosetransporters (GLUT1, GLUT3 and GLUT4) in B and T cells inpatients with type 2 diabetes mellitus managed with insulin.Material and methods. The study group consisted of 11 pa-tients with type 2 diabetes mellitus receiving intensive insulintreatment (4 injections daily). The control group comprised12 healthy individuals with no evidence of abnormal glucosemetabolism and no genetic predisposition for type 2 diabetesmellitus. We isolated lymphocytes and labelled them withmonoclonal murine CD19 and CD3 antibodies to determineB and T cell counts and with antibodies demonstratingexpression of specific glucose transporters. The sampleswere analysed by flow cytometry.

Results. The mean fluorescence intensity (MFI) for GLUT1and GLUT4 in B cells from diabetic patients was higher thanthat in the control group and the GLUT3 content was lower.The B cell GLUT3 content correlated with body mass indexvalues in diabetic patients and in healthy individuals(coefficients of correlation 0.80 and 0.83, respectively). InT cells, the mean MFI for the glucose transporters investigatedwas higher in the control group and in the case of GLUT3and GLUT4 the difference was statistically significant.Conclusions. Reduced levels of the highly efficient GLUT3and GLUT4 glucose transporters in T cells may be thecause of the reduced cell-mediated immunity. Type 2diabetes mellitus affects the quantitative contents ofglucose transporters in lymphocytes compared toindividuals without carbohydrate metabolism abnormalities.

Diabet Dośw i Klin 2008, 8, 4, 159–164key words: glucose transporters, lymphocytes, type 2diabetes mellitus, glucose transport

IntroductionGlucose is transported from the blood into the cells

by transmembrane transporting proteins encoded bytwo separate gene families. The first family is represen-ted by the so-called glucose co-transporters (SGLT, so-

dium/glucose co-transporters). These proteins transportglucose into the cells by means of active transport, aga-inst the concentration gradient. Six isoforms of glucoseco-transporters have so far been described (SGLT1––SGLT6) encoded by respective genes (SLC5A1––SCL5A6) [1]. The other family of glucose transportersis made up of GLUT proteins. Fourteen types of GLUTproteins have been described in humans (GLUT1––GLUT12, GLUT14 and HMIT) encoded by respectivegenes (SLC2A1–SLC2A14) [2]. These proteins transportglucose along the concentration gradient, by means offacilitated diffusion. Expression of the individual SLC2Agenes is tissue- and cell-specific [1]. They play an im-portant role in the maintenance of glucose homoeosta-sis in the body. GLUT1, for instance, is involved in thebasic glucose transport into the cells, while GLUT3 and

Address for correspondence:mgr inż. Agnieszka Sobczyk-KopciołZakład Biologii Ogólnej i ParazytologiiWarszawski Uniwersytet Medyczny, Instytut Biostrukturyul. Chałubińskiego 5, 02–004 WarszawaTel (+48 22) 621 26 07, Fax (+48 22) 628 53 50e-mail: [email protected]

Diabetologia Doświadczalna i Kliniczna 2008, 8, 4, 159–164Copyright © 2008 Via Medica, ISSN 1643–3165

Diabetologia Doświadczalna i Kliniczna 2008, Vol. 8, No. 4

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GLUT4 are responsible for insulin-stimulated transport [3].The net effect of these transporters is the uptake ofexcessive amounts of glucose in the blood by adipocy-tes and myocytes during periods of hyperglycaemia,resulting in the restoration of normoglycaemia.

Abnormalities of glucose metabolism caused by suchfactors as disturbed glucose transport into blood cellsimpair their function. Rat studies have shown that thereduced intensity of glucose transport into spleen lym-phocytes decreases their mitotic activity, which is mostlikely caused by underexpression of the gene that enco-des GLUT1 [4]. Expression of genes encoding glucosetransporters in humans is also affected by long-stan-ding hypoglycaemia. In vivo, long-standing hypoglyca-emia in humans increases GLUT4 levels in granulocytesby 73%, reduces GLUT1 by a similar value and slightlyrises the level of GLUT3. In monocytes, GLUT3 levelsincrease by 134% with no changes observed in the le-vels of GLUT1 or GLUT4. In lymphocytes, hypoglyca-emia has no effect on the expression of genes encodingglucose transporters [5].

Glucose metabolism and the physiological functionsof blood cells are also affected by protracted hypergly-caemia. In hyperglycaemia the increased level and con-formational changes of GLUT1 are observed in erythro-cyte membranes [6], which increases the energy of activa-tion necessary for transmembrane glucose transport [7]and reduces diffusion of glucose into the erythrocyte [8].Other consequences of long-standing hyperglycaemiainclude increased erythrocyte aggregation [9] and chan-ges in the lipid composition of the cell membrane [10].Fluctuations of glucose concentrations observed in pa-tients with type 2 diabetes mellitus affected peripheralblood lymphocyte function. Findings from in vivo stu-dies include: reduced activity of pyruvate dehydrogena-se [11], impairment of deoxy-D-glucose transport intothese cells and changes in glucose transporter levels inlymphocytes [12, 13]. Incubation of lymphocytes in non-physiological concentrations of glucose, namely in hy-poglycaemic and hyperglycaemic conditions, affectsdeoxy-D-glucose transport into peripheral blood lym-phocytes and the content of glucose transporters in thesecells. Particularly important abnormalities have beenfound in hyperglycaemia [14].

Granulocyte functions are also impaired in patients withtype 2 diabetes mellitus. Abnormal findings include incre-ased granulocyte aggregation [15] and reduced phago-cytic ability with respect to various pathogens [16].

The increased energy requirement in activated lympho-cytes results in increased glucose consumption [17, 18].Inactive T cells do not express insulin receptors, in con-trast to lymphocytes activated by phytohaemagglutinin[19] and in response to acute hyperglycaemia [20]. Ac-tivation of T cells and insulin receptor expression is also

triggered by cytokines and growth factors. Similar pro-cesses to those in lymphocytes have also been observ-ed in patients with ketoacidosis [21]. T cells activated byphytohaemagglutinin also express receptors for insulin-like growth factor-1 (IGF-1) and interleukin-2 [22, 23].

The aim of the study was to evaluate the presenceand contents of selected isoforms of glucose transpor-ters in B and T cells in patients with type 2 diabetesmellitus managed with insulin.

Material and methods

SubjectsThe study group consisted of 11 patients with type 2

diabetes mellitus receiving intensive insulin therapy(4 injections daily) hospitalised at the Clinic of Gastroen-terology and Metabolic Disorders, Medical University ofWarsaw, Poland. The control group was composed of12 healthy individuals with no abnormalities of glucosemetabolism and no genetic predisposition to type 2 dia-betes mellitus. These individuals had not taken anypharmacological agents during and for 3 months priorto the study. The detailed characteristics of the studysubjects are presented in Table 1.

The study was conducted in line with the ICH GCPguidelines, including the ethical principles laid down inthe Declaration of Helsinki (updated in Edinburgh), andwith the approval of the Bioethics Committee of the Me-dical University of Warsaw. The study subjects wereinformed of the purpose of observation and providedinformed consent to participate in the study.

Lymphocyte isolationFive millilitres of venous blood were collected after

an overnight fast into a heparin tube from each subject.The blood cells were isolated within 2 hours of bloodcollection. Lymphocytes were isolated by means of cen-trifugation in the gradient of Histopaque-1077 (Sigma).

Flow cytometryFollowing isolation, approximately 5 × 105 cells from

healthy individuals and patients were washed in 2 mL ofthe washing buffer into FACS (PBS without Mg2+ and Ca2+

with the addition of 2% FBS and 0.002% sodium azide) bycentrifugation (4°C, 250 g) and suspended in 100 µL of thebuffer. Samples prepared for a further procedure werekept in ice. In order to label B and T cells, they wereincubated with monoclonal murine antibodies, CD19 andCD3, respectively (Becton-Dickinson) conjugated with phy-coerythrin at 4°C for 30 minutes. Lymphocytes incubatedwith isotypic antibodies (Becton-Dickinson) served ascontrols. In order to achieve permeabilisation enabling


Agnieszka Sobczyk-Kopcioł et al. Expression of selected glucose transporters in peripheral blood lymphocytes

antibodies to the intracellular domain of glucose transpor-ters to enter the cytosol the cells were incubated for 10minutes with 0.5 mL Perm 2 (Becton-Dickinson). The blo-od cells were then washed in the buffer into FACS andincubated for 30 minutes with 2 µL polyclonal rabbit anti-bodies to GLUT1, GLUT3 and GLUT4 (Chemicon) ata 1:50 dilution. Instead of antibodies, 5 µL of polyclonalrabbit serum (Dako) were added to the control sample.The cells were then washed in the buffer into FACS andincubated with the secondary antibody (Dako) labelledwith FITC at a 1:40 dilution at 4°C for 30 minutes in thedark. After the incubation 2 mL of the washing buffer intoFACS were added to each of the samples, the resultingsuspensions were centrifuged (4°C, 250 g), the superna-tant was removed and 0.5 mL of the washing buffer intoFACS and 1% formaldehyde were added.

The samples were analysed using the FACS Caliburflow cytometer (Becton-Dickinson) fitted with an argonlaser (wavelength, 488 nm) using the CellQuest software.

Presentation of results andstatistical analysis

The results were presented as the mean fluorescen-ce intensity (MFI), a product of the number of cells inwhich glucose transporters were labelled and the inten-sity of fluorescence of antibodies labelling these gluco-se transporters. The statistical analysis was performed

with the use of Statistica 7 and was based on thet-Student test (95% CI).

ResultsMean fluorescence intensity for GLUT1 in B cells

from patients with type 2 diabetes mellitus was higherthan that in the control group (1461.5 ± 352.5 vs.958.4 ± 474.3). The level of GLUT3 was higher inhealthy individuals compared to diabetics (833.3 ±± 582.6 vs. 599.17 ± 818.1). Cytoplasm levels ofGLUT4 were higher in diabetics compared to healthyvolunteers (513.6 ± 792.8 vs. 408.0 ± 210.6) (Table 2,Figure 1–3). B cell GLUT3 levels correlated with bodymass index (BMI) values in diabetic patients and in heal-thy individuals (coefficients of correlation 0.80 and 0.83,respectively).

In T cells, the mean value of MFI for GLUT1 was hi-gher in the control group compared to diabetics (8101.04± 4464.7 vs. 6528.4 ± 962.6). There was a significantdifference in the levels of GLUT3 and GLUT4 in T cellsbetween the study group and healthy volunteers (2306 ±± 1732.5 vs. 6455.6 ± 2831.8 for GLUT3 and 1935.1 ±± 1935.1 vs. 7013.4 ± 3905.5 for GLUT4) (Table 3, Figu-re 4–6). In this case, GLUT4 levels in T cells correlatedwith BMI only in the control group.

Table 1. Study subject characteristics

Patients (n = 11) Healthy individuals (n = 11)

Male:Female ratio 5:6 6:6Age (years) 60 ± 12.26 55 ± 8.56Duration of the disease (years) 12 ± 5.87 Not applicableBody mass index [kg/m2] 27.03 ± 1.84 24.88 ± 3.03Fasting glucose [mg%] 124.4 ± 21.6 85.5 ± 5.42C-peptide [ng/mL] 1.9 ± 0.97 –

Table 2. Mean fluorescence intensity of glucose transporters in B cells

Glucose transporters Patients Controls P

GLUT1 1461.522 ± 352.5 958.4038 ± 474.3 0.082029GLUT3 599.1795 ± 818.1 833.2598 ± 582.6 0.494582GLUT4 513.6778 ± 792.8 408.0243 ± 210.6 0.719591

Table 3. Mean fluorescence intensity of glucose transporters in T cells; *statistically significant

Glucose transporters Patients Controls P

GLUT1 6528.4 ± 962.6 8101.04 ± 4464.7 0.580529GLUT3 2306 ± 1732.5 6455.6 ± 2831.8 0.023380*GLUT4 1935.1 ± 1935.1 7013.4 ± 3905.5 0.030584*



Figure 1. Mean fluorescence intensity for GLUT1 in B cells;SE — standard error



Figure 4. Mean fluorescence intensity for GLUT1 in T cells;SE — standard error




Agnieszka Sobczyk-Kopcioł et al. Expression of selected glucose transporters in peripheral blood lymphocytes

DiscussionIn vitro studies investigating the effects of increasing

concentration of glucose on GLUT4 expression on thesurface of T cells have demonstrated a positive correla-tion between the increased level of this glucose transpor-ter and the concentration of glucose. A positive correla-tion has also been shown between the concentration ofglucose and the concentrations of proinflammatory cyto-kines released by T cells and expression of insulin andIGF-1 receptors [24]. In patients with type 2 diabetesmellitus an increased expression of GLUT4 on the surfa-ce of lymphocytes has been observed [25, 26].

The level and regulation of expression of glucose trans-porters in the muscle and adipose tissues has been a topicof numerous studies [27–30]. In conditions of insulin resi-stance, expression of the gene encoding GLUT4 is regula-ted in a different manner in adipose cells and myocytes.

GLUT4 mRNA and GLUT4 protein are reduced in theadipose tissue of obese individuals and patients with type 2diabetes mellitus, while no quantitative changes in theseparameters are observed in the myocytes [31, 32].

Cell culture studies have shown that a high level ofinsulin, rather than its lack, reduces the level of GLUT4mRNA. Animal studies have demonstrated that selectiveunderexpression of GLUT4 in adipocytes results in glu-cose intolerance and hyperinsulinaemia [33].

In studies investigating glucose transporter expressionon the surface of B and T cells have shown that in thebasic state, only B cells increase the levels of GLUT3 andGLUT4 in response to insulin. The expression of GLUT3 inB cells correlated with BMI and fasting insulin levels [34].

At physiological concentrations insulin stimulates cyto-toxicity of lymphocytes [35]. Reduced levels of the highlyefficient GLUT3 and GLUT4 glucose transporters in T cellsmay be the cause of the reduced cell-mediated immunity.

ConclusionsType 2 diabetes mellitus affects the quantity of gluco-

se transporters in lymphocytes compared to individualswithout carbohydrate metabolism abnormalities. The dif-ferent expression of the selected GLUT receptors onsubpopulations of T and B cells may be associated withimpaired cell-mediated and humoral immunity observedin the course of diabetes mellitus.

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