Genetic Modulation of the GLUT1 Transporter Expression-Potential Relevance in Complex Diseases.

The human GLUT1 (SLC2A1) membrane protein is the key glucose transporter in numerous cell types, including red cells, kidney, and blood-brain barrier cells. The expression level of this protein has a role in several diseases, including cancer and Alzheimer's disease. In this work, to investigate a potential genetic modulation of the GLUT1 expression level, the protein level was measured in red cell membranes by flow cytometry, and the genetic background was analyzed by qPCR and luciferase assays. We found significant associations between red cell GLUT1 levels and four single nucleotide polymorphisms (SNP) in the coding SLC2A1 gene, that in individuals with the minor alleles of rs841848, rs1385129, and rs11537641 had increased, while those having the variant rs841847 had decreased erythrocyte GLUT1 levels. In the luciferase reporter studies performed in HEK-293T and HepG2 cells, a similar SNP-dependent modulation was observed, and lower glucose, serum, and hypoxic condition had variable, cell- and SNP-specific effects on luciferase expression. These results should contribute to a more detailed understanding of the genetic background of membrane GLUT1 expression and its potential role in associated diseases.

Potential role of the ABCG2-Q141K polymorphism in type 2 diabetes.

Type 2 diabetes mellitus (T2DM) is a complex metabolic disease and variations in multispecific membrane transporter functions may affect T2DM development, complications or treatment. In this work we have analyzed the potential effects of a major polymorphism, the Q141K variant of the ABCG2 transporter in T2DM. The ABCG2 protein is a multispecific xeno- and endobiotic transporter, affecting drug metabolism and playing a key role in uric acid extrusion. The ABCG2-Q141K variant, with reduced expression level and function, is present in 15-35% of individuals, depending on the genetic background of the population, and has been shown to significantly affect gout development. Several other diseases, including hypertension, chronic renal failure, and T2DM have also been reported to be associated with high serum uric acid levels, suggesting that ABCG2 may also play a role in these conditions. In this work we have compared relatively small cohorts (n = 203) of T2DM patients (n = 99) and healthy (n = 104) individuals regarding the major laboratory indicators of T2DM and determined the presence of the SNP rs2231142 (C421A), resulting the ABCG2-Q141K protein variant. We found significantly higher blood glucose and HbA1c levels in the T2DM patients carrying the ABCG2-Q141K variant. These findings may emphasize the potential metabolic role of ABCG2 in T2DM and indicate that further research should explore how prevention and treatment of this disease may be affected by the frequent polymorphism of ABCG2.

Alterations in erythrocyte membrane transporter expression levels in type 2 diabetic patients.

Type 2 diabetes mellitus (T2DM) is one of the most common multifactorial diseases and several membrane transporters are involved in its development, complications and treatment. We have recently developed a flow-cytometry assay panel for the quantitative determination of red cell membrane protein levels with potential relevance in diseases. Here we report a detailed phenotypic analysis of a medium scale, clinically based study on the expression of T2DM-related membrane proteins, the GLUT1, GLUT3, MCT1, URAT1, ABCA1, ABCG2 and the PMCA4 transporters in erythrocytes. By comparing age-matched control subjects and three groups of T2DM patients (recently diagnosed, successfully managed, and patients with disease-related complications), we found significant differences in the membrane expression levels of the transporters in these groups. This is a first detailed analysis of T2DM related alterations in erythrocyte membrane transporter protein levels, and the results suggest significant changes in some of the transporter expression levels in various patient groups. By performing a further, more detailed analysis of the clinical and molecular biology parameters, these data may serve as a basis of establishing new, personalized diagnostic markers helping the prevention and treatment of type 2 diabetes.

Stimulus-triggered enhancement of chilling tolerance in zebrafish embryos.

BACKGROUND: Cryopreservation of zebrafish embryos is still an unsolved problem despite market demand and massive efforts to preserve genetic variation among numerous existing lines. Chilled storage of embryos might be a step towards developing successful cryopreservation, but no methods to date have worked. METHODS: In the present study, we applied a novel strategy to improve the chilling tolerance of zebrafish embryos by introducing a preconditioning hydrostatic pressure treatment to the embryos. In our experiments, 26-somites and Prim-5 stage zebrafish embryos were chilled at 0°C for 24 hours after preconditioning. Embryo survival rate, ability to reach maturation and fertilizing capacity were tested. RESULTS: Our results indicate that applied preconditioning technology made it possible for the chilled embryos to develop normally until maturity, and to produce healthy offspring as normal, thus passing on their genetic material successfully. Treated embryos had a significantly higher survival and better developmental rate, moreover the treated group had a higher ratio of normal morphology during continued development. While all controls from chilled embryos died by 30 day-post-fertilization, the treated group reached maturity (~90-120 days) and were able to reproduce, resulting in offspring in expected quantity and quality. CONCLUSIONS: Based on our results, we conclude that the preconditioning technology represents a significant improvement in zebrafish embryo chilling tolerance, thus enabling a long-time survival. Furthermore, as embryonic development is arrested during chilled storage this technology also provides a solution to synchronize or delay the development.

Cell surface membrane proteins as personalized biomarkers: where we stand and where we are headed.

Personalized medicine requires the development of a wide array of biomarker diagnostic assays, reflecting individual variations and thus allowing tailored therapeutic interventions. Membrane proteins comprise approximately 30% of total human proteins; they play a key role in various physiological functions and pathological conditions, although, currently, only a limited number of membrane proteins are applied as biomarkers. In many normal tissues, cell surface membrane proteins are not easily accessible for diagnostic sampling, and tumor-derived membrane preparations - while serving as potential tumor biomarkers - may not reflect physiological protein expression. In addition to post-translational modifications, which may include glycosylation, phosphorylation and lipid modifications, the trafficking of membrane proteins is also regulated. Moreover, a tight cellular quality control monitors membrane protein maturation, and continuous removal and reinsertion, involving special signaling systems, occurs in many cases. However, cell surface membrane proteins already serve as valuable prognostic and predicative biomarkers, for example, in hematological and immunological diseases, by the determination of the cluster of differentiation markers. In this review, we demonstrate the relevance of cell surface membrane biomarkers in various diseases and call attention to the potential application of red blood cell (erythrocyte) membrane proteins in this regard. Surprisingly, red blood cells express hundreds of membrane proteins, which seem to reflect a general genetic and regulatory background, and may serve as relatively stable and easily accessible personalized membrane biomarkers. Quantitative membrane protein detection in red blood cells by flow cytometry may bring a breakthrough in this regard.

Peripheral blood-derived autologous stem cell therapy for the treatment of patients with late-stage peripheral artery disease-results of the short- and long-term follow-up.

BACKGROUND AIMS: Regeneration of the occluded peripheral arteries by autologous stem cell therapy is an emerging treatment modality for no-option patients with peripheral artery disease (PAD). The purpose of this study was to assess safety and efficacy of in vitro-expanded, peripheral blood-derived, autologous stem cells (VesCell) in no-option patients with PAD. METHODS: A phase II, open-label, randomized clinical study was performed on 20 patients to investigate the safety and efficacy of VesCell therapy at 1 and 3 months of follow-up. The long-term (2 years) efficacy of the therapy was also evaluated. RESULTS: No side effects of VesCell therapy were found. During the 3 month follow-up in the control group, one death occurred and six major amputations were performed; in the treated group, there were no deaths or major amputations. The difference of limb loss is significant between the two groups. At 2-year follow-up in the control group, two deaths and six major amputations occurred; in the treated group, there were three major amputations. At 3-month follow-up, the change in hemodynamic parameters showed a significant increase in the treated group over the control group; in the treated group, further improvement was detected at 2 years. As the result of the VesCell treatment, change in pain score, wound healing and walking ability test showed an improvement compared with the control group; at 2 years, incremental improvement was observed. CONCLUSIONS: Peripheral blood-derived, in vitro-expanded autologous angiogenic precursor therapy appears to be a safe, promising and effective adjuvant therapy for PAD patients.

Functional ABCG1 expression induces apoptosis in macrophages and other cell types.

The expression of the ATP-binding cassette transporter ABCG1 is greatly increased in macrophages by cholesterol loading via the activation of the nuclear receptor LXR. Several recent studies demonstrated that ABCG1 expression is associated with increased cholesterol efflux from macrophages to high-density lipoprotein, suggesting an atheroprotective role for this protein. Our present study uncovers an as yet not described cellular function of ABCG1. Here we demonstrate that elevated expression of human ABCG1 is associated with apoptotic cell death in macrophages and also in other cell types. We found that overexpression of the wild type protein results in phosphatidyl serine (PS) translocation, caspase 3 activation, and subsequent cell death, whereas neither the inactive mutant variant of ABCG1 (ABCG1K124M) nor the ABCG2 multidrug transporter had such effect. Induction of ABCG1 expression by LXR activation in Thp1 cells and in human monocyte-derived macrophages was accompanied by a significant increase in the number of apoptotic cells. Thyroxin and benzamil, previously identified inhibitors of ABCG1 function, selectively prevented ABCG1-promoted apoptosis in transfected cells as well as in LXR-induced macrophages. Collectively, our results suggest a causative relationship between ABCG1 function and apoptotic cell death, and may offer new insights into the role of ABCG1 in atherogenesis.

Tyrosine kinase inhibitor resistance in cancer: role of ABC multidrug transporters.

Recent antitumor drug research has seen the development of a large variety of tyrosine kinase inhibitors (TKIs) with increasing specificity and selectivity. These are highly promising agents for specific inhibition of malignant cell growth and metastasis. However, their therapeutic potential also depends on access to their intracellular targets, which may be significantly affected by certain ABC membrane transporters. It has been recently shown that several human multidrug transporter ABC proteins interact with specific TKIs, and the ABCG2 transporter has an especially high affinity for some of these kinase inhibitors. These results indicate that multidrug resistance protein modulation by TKIs may be an important factor in the treatment of cancer patients; moreover, the extrusion of TKIs by multidrug transporters may result in tumor cell TKI resistance. Interaction with multidrug resistance ABC transporters may also significantly modify the pharmacokinetics and toxicity of TKIs in patients.

Functional expression and characterization of the human ABCG1 and ABCG4 proteins: indications for heterodimerization.

The closely related human ABC half-transporters, ABCG1 and ABCG4, have been suggested to play an important role in cellular lipid/sterol regulation but no experimental data for their expression or function are available. We expressed ABCG1 and ABCG4 and their catalytic site mutant variants in insect cells, generated specific antibodies, and analyzed their function in isolated membrane preparations. ABCG1 had a high basal ATPase activity, further stimulated by lipophilic cations and significantly inhibited by cyclosporin A, thyroxine or benzamil. ABCG4 had a lower basal ATPase activity which was not modulated by any of the tested compounds. The catalytic site (K-M) mutants had no ATPase activity. Since dimerization is a requirement for half-transporters, we suggest that both ABCG1 and ABCG4 function as homodimers. Importantly, we also found that co-expression of the ABCG4-KM mutant selectively abolished the ATPase activity of the ABCG1 and therefore they most probably also heterodimerize. The heterologous expression, specific recognition, and functional characterization of these transporters should help to delineate their physiological role and mechanism of action.

The role of ABC transporters in drug resistance, metabolism and toxicity.

ATP Binding Cassette (ABC) transporters form a special family of membrane proteins, characterized by homologous ATP-binding, and large, multispanning transmembrane domains. Several members of this family are primary active transporters, which significantly modulate the absorption, metabolism, cellular effectivity and toxicity of pharmacological agents. This review provides a general overview of the human ABC transporters, their expression, localization and basic mechanism of action. Then we shortly deal with the human ABC transporters as targets of therapeutic interventions in medicine, including cancer drug resistance, lipid and other metabolic disorders, and even gene therapy applications. We place a special emphasis on the three major groups of ABC transporters involved in cancer multidrug resistance (MDR). These are the classical P-glycoprotein (MDR1, ABCB1), the multidrug resistance associated proteins (MRPs, in the ABCC subfamily), and the ABCG2 protein, an ABC half-transporter. All these proteins catalyze an ATP-dependent active transport of chemically unrelated compounds, including anticancer drugs. MDR1 (P-glycoprotein) and ABCG2 preferentially extrude large hydrophobic, positively charged molecules, while the members of the MRP family can extrude both hydrophobic uncharged molecules and water-soluble anionic compounds. Based on the physiological expression and role of these transporters, we provide examples for their role in Absorption-Distribution-Metabolism-Excretion (ADME) and toxicology, and describe several basic assays which can be applied for screening drug interactions with ABC transporters in the course of drug research and development.