Research areas, specific techniques and reagents of participating labs

Bulgaria

Dimiter Dimitrov

Research areas

• main research areas: obese type 2 diabetes mellitus patients with the components of the metabolic ( X ) syndrome and the role of leptin in the pathogenesis of insulin resistance and its complications

Specific techniques used

• our lab invesigates leptin ( RIA ), IRI ( RIA ), lipid and lipoprotein status, ultrasound and CT measurement of abdominal fat mass. We plan to investigate leptin receptors and additional factors involved in insulin resistance

Specific reagents available

• endocrinological reagents

Czech Republic

Bela Bendlova

Research areas

• identification of specific gene markers of Reaven´s matabolic syndrome (NIDDM, obesity and polycystic ovarian syndrome) in Czech population
• correlations of DNA polymorphisms with clinical and metabolic parameters of NIDDM patient databank including the genetic epidemiology of MODY gene mutations

Specific techniques used

• TGGE (temperature gradient gel elfo - Biometra), DNA sequencing (ALF Express-Pharmacia)

Specific reagents available

• determination of a wide range of hormones (including some infrequrently determined steroids and their metabolites - e.g. allopregnanolone, pregnanolone, pregnanolone sulfate, epitestosterone etc.)

Jaroslav Vesely

Research areas

• a laboratory insulin-resistant animal model - the Koletsky rats - has been explored and characterized. Lipoproteins in distinct groups of patients and laboratory animals have been studied. A study of a novel class of synthetic trisubstituted purine inhibitors of insulin receptor tyrosine-kinase has been performed by us. We plan to concentrate on the following topics:
• molecular mechanisms of insulin resistance and dyslipidemias and of their clinical manifestations
• molecular biological (genetic) basis of carbohydrate and lipid metabolism defects, incl. a search for their candidate genes
• relationships between carbohydrate and lipid metabolic fenotypes and health status in different population groups, incl. young people
• utilisation of new screening and laboratory parameters in clinical diagnosis of the disturbances

Danemark

Aase Handberg

Research areas

• cultures of differentiated human skeletal muscle cells
• effect of diabetes and age on glucose transporter level and localization in human muscle
• muscle fiber type, insulin action, obesity and age
• receptor phosphorylation and dephosphorylation
• pathogenesis of NIDDM

Christine Reynet

Research areas

• insulin/IGF-I signaling and glucose metabolism regulation in adult skeletal muscle cells
• insulin signaling and resistance in cultured human skeletal muscle cells from control and NIDDM subjects
• regulation of glucose transport and GLUT-4 trafficking using tagged glucose transporters

Specific techniques used

• molecular biology: DNA cloning, cell transfection, Northern and Western blot analyses, RT-PCR, reporter gene assay, etc.
• cell biology: a variety of bioassays, immunocytochemistry, cell imaging, etc.

France

Emmanuel Van Obberghen

Research areas

• signaling by insulin receptors, IGF-1 receptors,VEGF receptors
• molecular and cellular defects of insulin resistance
• search for genes involved in diabetes type 2 and obesity
• VEGF and diabetic microvascular complications
• crosstalk between insulin/IGF-1 signaling and signaling by integrins

Specific techniques used

• measurement of biological responses relating to metabolism, growth and differentiation
• expression of mutated molecules(stable and transient transfections)
• yeast two and three hybrid system
• cDNA microarrays
• suppression subtractive hybridization

Germany

Jurgen Eckel

Research areas

• insulin action and signalling in cardiac muscle
• mechanisms of insulin resistance
• molecular pathways of GLUT4 translocation
• molecular pharmacology of antidiabetic drugs

Greece

George Dimitriadis, Effie Tsilibary

Research areas

• insulin resistance on skeletal muscle and adipose tissue in humans (type 2 diabetes, effects of anti-insulin hormones - thyroxine - on carbohydrate metabolism)
• glucose metabolism in human blood cells (monocytes, erythrocytes)
• protein and lipid metabolism in diabetes
• measurements of expression of cell surface intregrin receptors in blood cells (extracellular matrix-cell interactions, integrin receptors, integrin-mediated signalling)
• effects of insulin secretagogues and insulin sensitizers on blood glucose control in patients with type 2 diabetes
• development of diagnostic methods for the early prediction of diabetic nephropathy

Specific techniques used

• primary cultures of skeletal muscle cells
• isolation of monocytes, lymphocytes (with monoclonal antibodies), erythrocytes
• euglycemic-hyperinsulinemic clamps, arterio-venous differences in subcutaneous abdominal adipose tissue and forearm skeletal muscles, microdialysis technique
• measurements of blood flow in subcutaneous abdominal adipose tissue (with 133Xe) and forearm skeletal muscles (plethysmography)
• Artificial Endocrine Pancreas (Biostator^R)

Specific reagents available

• reagents related to cell culturing, isolation of blood cells and integrin receptors

Hungary

Peter Csermely

Research areas

• changes of molecular chaperone action in diabetes
• changes of redox status in diabetes

Specific techniques used

• chaperone assays

Specific reagents available

• chaperone antibodies, cDNA-s, constructs (GST, MDH, GFP etc.), mutants, RNA probes etc.

Gyorgy Jermendy

Research areas

• Population-based clinical investigation is planned to get new information about the details of metabolic X-syndrome in Hungarian population. Analysis of the etiology of insulin resistance, obesity, impaired glucose tolerance or non-insulin-dependent diabetes mellitus in connection with the clinical picture of suspected early atherosclerosis are planned. Major methods: serum insulin fasting, post-loading, serum C-peptide fasting, post loading, serum homocysteine, serum vitamin B12, B6, clinical investigation for detecting early atherosclerosis (duplex scan, exercise testing)

Ireland

John Nolan

Research areas

• insulin resistance in young and elderly patiens
• developing a better clinical classification of diabetes
• establishment of immune markers detecting latent autoimmune diabetes in adults (LADA)
• insulin resistance in obesity
• molecular markers of obesity and its treatment

Specific techniques used

• muscle biopsy, glucose clamp, signaling assays, exercise physiology

Israel

Shlomo Sasson

Research areas

• autoregulation of glucose transport in skeletal muscle cells and adipose cells
• role of lipoxygenase in the regulation of glucose transport in vascular endothelial and smooth muscle cells
• effects reactive oxygen species and antioxidants on the glucose transport system
• development of novel antihyperglycemic drugs

Specific techniques used

• biotinylation method to measure cell surface proteins (e.g., glucose transporters).
• competitive PCR technique for GLUT-1 mRNA
• various polyclonal antibodies against rat glucose transporters.

Italy

Francesco Giorgino

Research areas

• glucose transporter regulatory mechanisms
• insulin signaling in diabetic skeletal muscle and myocardium
• effects of steroids on insulin action
• functional analysis of PI 3-kinase
• molecular mechanisms of IGF-mediated cell growth and differentiation

Specific techniques used

• techniques to study protein-protein interactions
• adenoviral gene transfer techniques

Specific reagents available

• antibodies to insulin signaling proteins and glucose transporters
• glucose transporter cDNAs
• recombinant glucose transporter fragments

Lithuania

Valentinas Matulevicius

Research areas

• insulin resistance
• polycystic ovary syndrome
• androgen-related insulin resistance
• clinical aspects and management
• connections between obesity, diabetes and physical activity

Vaidotas Urbanavicius

Research areas

• mechanisms of insuline resistance in type 2 diabetes mellitus and obesity
• postprandial glucose and lipid metabolism in type 2 diabetes and obesity
• glucose metabolism in red blood cells

Specific techniques used

• investigation of insulin resistance and postprandial glucose and lipid metabolism in vivo
• red blood cell glucose metabolism in vitro

The Netherlands

Ton Maassen

Research areas

• insulin receptor mutation analysis
• insulin signal transduction
• adipocytes: signal transduction
• mitochondria and their contribution to glucose homeostasis
• gene variants and their association with changes in glucose homeostasis

Norway

Jorgen Jensen

Research areas

• effect of stress hormones (adrenaline and cortisol) on insulin sensitivity in skeletal muscle
• effect of contractile activity on insulin sensitivity in skeletal muscle

Specific techniques used

• We have a system where we can stimulate skeletal muscles in vitro and study basic mechanisms for how contractile activity and stress hormones influence insulin sensitivity. Furthermore, we have a rat model for infusion of stress hormones via osmotic mini-pumps combined with blood sampling from chronic catheters. In vitro, we measure glucose transport (3H-3-O-methyl glucose), 3H-2 deoxyglucose uptake, and glycogen synthesis (14C-glucose) in skeletal muscle. I frozen tissue we measure glycogen concentration and different metabolites biochemically. Activation of glycogen synthase and glycogen phosphorylase are measured radiochemically. We also do Western blot to study different proteins in the insulin signalling pathway.

Arild Rustan

Research areas

• n-3 fatty acids (n-3 PUFA) and 3-thia fatty acids (e.g. TTA): interplay between glucose and lipid metabolism in cultured human adult skeletal muscle cells from normal subjects, relatives and NIDDM patients
• effects of n-3 PUFA and TTA on regulation of lipid metabolism, leptin production and leptin-receptor expression in human adipocytes and 3T3-L1 adipocytes
• interaction between the effect of insulin, stress related hormones and TTA on fatty acid oxidation and triacylgycerol secretion by human liver cells (THLE cells)

Specific techniques used

• isolation and culturing of human skeletal muscle cells (methods obtained from PhD stud. Michael Gaster, Denmark)
• measurement of glucose metabolism using labeled precursors: glucose uptake, glycogen synthesis and glucose oxidation. Measurement of fatty acid metabolism also by use of labeled precursors: fatty acid oxidation, triacylglycerol synthesis and lipolysis, synthesis of other complex lipids
• measurement of fatty acid composition of lipids, certain enzymes and receptors involved in glucose and lipid metabolism on both gene and protein levels (in collabotation with other groups): PPARs, CPT-I and CPT-II, FAO, leptin receptor, LDL-receptor, FABPs, GLUT4

Specific reagents available

• 3-thia fatty acids, other hypolipidemic and hyperlipidemic fatty acid derivatives (all from prof. Rolf K. Berge, Norway)

Romania

Maya Simionescu (Doina Popov)

Research areas

• advanced glycation endproducts of proteins: transport of reversibly and irreversibly glycated albumin; the effects of irreversibly glycated proteins on low density lipoproteins; the occurrence of the protein “carbonyl stress”
• oxidant/antioxidant imbalance in diabetes: molecular mechanisms involved in the protective effect of natural antioxidants to reduce the oxidative stress in diabetes and lipoprotein peroxidation in diabetes-induced accelerated atherosclerosis
• endothelial dysfunction in diabetes: the mechanisms underlying the alteration of the vascuIar reactivity of resistance arteries to different agonists; NO depletion/reduced synthesis or availability
• accelerated atherosclerosis as the main complication in diabetes
• cellular and molecular events leading to diabetes and its main complication - accelerated atherosclerosis: the use of the hyperlipemic-hyperglycemic hamster; an original animal model to study the modifications of plasma components and of the tissues most affected by these diseases: heart, cardiac valves, retina, kidney, blood vessels; biochemical alterations of the vascular wall and circulating cells; structural, functional and biochemical studies of the resistance arteries of hyperlipemic hyperglycemic hamsters
• in vitro models for drug testing
• the effect of drugs on cultured vascular cells (endothelial, smooth muscle cells) incubated with various modified lipoproteins; an attempt to simulate the deleterious in vivo conditions occurring in diabetes and atherosclerosis, and their modulation or regression under the influence of drugs

specific techniques used

• experimental models of diabetes and/or atherosclerosis: hyperglycemic and/or hyperlipemic hamsters
• electron microscopy
• fluorescence microscopy
• cell culture
• immunoassays
• spectrofluorimetry
• radioisotope labeling
• autoradiography for light and electron microscopy.
• HPLC
• Polymerase Chain Reaction
• myography of small vessels

specific reagents available

• cultured cells: endothelial cells (from human placenta, bovine aorta, pulmonary artery and heart valves), transfected endothelial cells expressing different receptors; smooth muscle cells (from human and other species)
• AGE-albumin
• human lipoproteins from normal and diabetic subjects
• glycated lipoproteins

Elena M.A. Ganea

Research areas

• nonenzymatic posttranslational modifications of proteins, such as: glycation, carbamylation (KNCO binding), aldehyde binding (MDA)
• in vitro glycation of proteins: lens proteins, myelin basic protein, myelin associated protein, insulin and enzymes
• chaperone-like activity of a -crystallin in protecting proteins and enzymes against changes induced by various posttranslational modification, enzyme renaturation

specific techniques used

• lens protein separation and purification
• myelin basic protein and myelin associated glycoprotein purification
• structural changes induced by glycation are estimated by absorption and fluorescence spectra, SDS-PAGE and immunochemical methods
• specific rabbit immune sera preparation against the native proteins
• alteration of the biological activity induced by glycation and other posttranslational modifications, estimated by measuring enzymatic activity

• a -crystallin complex formation assayed by HPLC and immunoblotting

specific reagents available

• specific immune sera: anti-soluble lens crystallins, anti-a -crystallin, anti-MBP, anti-G6PD.

Dan Cheta

Research areas

• epidemiology (diabetes, obesity, lipid disorders)
• treatment of type 2 diabetes by classical and new methods
• diabetes complications (neuropathy, retinopathy, nephropathy)
• ageing and nutrition
• ageing and metabolism

Specific techniques used

• plasma lipid profile and metabolic status determination
• ophtalmology, nephrology in diabetes
• neuroelectrophysiological techniques

Specific reagents available

• bioinformatic, nephrology, ophtalmology and electrophysiology techniques

Slovakia

Iwar Klimes, Elena Sebokova

Research areas

• Role of lipids in the development of insulin resistance
• regulation of fatty acid oxidation in relation to insulin action in animals with diet- induced or hereditary fixed insulin resistance after dietary and pharmacological interventions leading to decreased availability of triglycerides and fatty acids
• elucidation of the role of malonyl-CoA and active forms of long chain fatty acids (LCFA) participating in regulation of fatty acid oxidation for the development of insulin resistance
• molecular mechanism of fatty acid desaturation responsible for altered fatty acid and lipid composition in insulin resistance
• regulation of transcription factors PPARs and SREBP involved in lipid and glucose metabolism in hypertriglyceridemia and insulin resistance
• role of leptin in insulin resistance and hypertriglyceridemia

Specific techniques used

• measurement of insulin action in vivo and glucose turnover studies by euglycemic hyperinsulinemic clamp
• analysis of fatty acids and lipids in blood and tissues by gas chromatography
• computer assisted direct measurement of blood pressure in conscious freely moving rats
• Northern and Western blot techniques for measurement of specific proteins and signalling molecules
• PCR and RT PCR techniques for measurements of expression for various enzymes of lipid and glucose metabolism, transcription factors or leptin

Slovenia

Robert Zorec, Marjan Kordas, Zoran Grubic

Research areas

• molecular cell physiology
• molecular mechanisms of regulated exocytosis and endocytosis
• insulin- and ATP-dependent dynamics of plasma membrane in cultured adipocytes
• changes in membrane surface area using FMI-43 fluorescent plasma membrane marker and confocal microscopy
• patch-clamp lock-in-amplifier for admittance measurements in small and large cells
• immunocytochemistry of SNARE proteins, GTP-binding proteins, CAPS
• monitoring vesicular trafficking with EGFP constructs and confocal microscopy

Specific techniques used

• single cell electrophysiology measurements of membrane capacitance
• standard patch-clamp measurements
• measurements of cytosolic calcium (photometry and imaging)
• caged calcium photolysis
• amperometry
• confocal microscopy
• immunocytochemistry
• microinjection techniques
• electroporation
• transfection, lipofection
• molecular biology, EGFP fusion constructs
• primary cell cultures and clonal cell culture facilities
• primary culture of noninnervated human muscle
• primary culture of in vitro innervated human muscle (rat-human and human-human co-cultures)
• in situ hybridization
• Western blotting

Specific reagents

• EGFP fusion constructs
• SNARE protein antibodies

Spain

Margarita Lorenzo

Research areas

• regulation of glucose uptake and GLUT4/1 gene expression in brown adipocytes
• insulin/IGF-I signaling in brown adipocytes and skeletal muscle cells
• apoptosis in brown adipocytes and skeletal muscle cells

Specific techniques used:

• primary cultures and cell line cultures
• ligand binding
• IR autophosphorylation assays, PI3-kinase activity, Western-blotting and immunoprecipitations
• transfections, CAT-assay, gel-shift assays
• RNA extraction, Northern-blot, nuclear run-on
• apoptosis and DNA content by flow cytometry, DNA fragmentation
• immunofluorescence and confocal microscopy

Specific reagents available:

• cDNAs, eukaryotic expression vectors, antibodies and chemical inhibitors related to the above techniques

Isabel Varela-Nieto

Research areas

• insulin lipidic mediators: GPI, IPG
• regulation of GPI-specific phospholipase activity by insulin
• novel biological sources for IPG
• cross-talk between PI-3-K and GPI signalling pathways

Antonio Zorzano

Research areas

• search of new molecules for glucose transport up-regulation
• differential expression of muscle genes in Type II diabetes mellitus
• differential expression of muscle genes in obesity
• search of new targets for the treatment of insulin resistance
• signalling events in muscle cell differentiation

Specific techniques used

• culture of muscle and adipose cells
• expression of proteins in Xenopus oocytes
• immunofluorescence assays
• plasma membrane lawn techniques
• subcellular membrane fractionation in adipose cells and in skeletal muscle
• immunogold assays
• freeze-etching analysis
• vesicle immunoisolation and immunoprecipitation assays
• production of adenovirus
• quantitative RT-PCR
• GLUT4 vesicle immunoisolation

Specific reagents available

• GLUT1 gene promoter constructs
• GLUT4 gene promoter constructs

Sweden

Jan Eriksson

Research areas

• mechanisms of insulin resistance in type 2 diabetes and obesity
• role of insulin resistance in the development of diabetic complications
• environmental factors causing insulin resistance
• fat cell metabolism

Juleen R. Zierath

Research areas

• Molecular mechanism for insulin and contraction-induced signal transduction in human skeletal muscle (effects of diet, ageing and environmental effects)

Specific reagents available

• human muscle biopsies from resting and exercising muscles

Switzerland

Jean-Louis Carpentier

Research areas

• trafficking of the insulin and leptin receptors
• cross-talk between the insulin and leptin receptors with emphasis on their traffick
• extension of these studies to include glucose transporters

Thomas Trueb

Research areas

• indentification and characterization of gene products interacting with known components of the insulin receptor signaling pathway (IRS-1, IRS-2, etc.)
• modulation of IRS protein-insulin receptor interactions
• identifying molecular determinants that target signaling components (like IRS-1) to a distinct subcellular compartment

Specific techniques used and reagents available

• a new adenovirus technique has been developed to produce recombinant adenovirus by Cre/Lox recombination in E. coli for easy selection of positive clones, than in HEK 293 cells to produce infectious adenoviri

Turkey

Yusuf Ozturk

Research areas

• calcium and calmodulin in experimental diabetes
• effect of adenosine in diabetes

UK

Ken Siddle

Research areas

• structure and function of receptors for insulin and IGFs, including determinants of ligand binding specificity and mechanism of ligand-induced receptor activation
• intracellular signalling by insulin receptors and IGF receptors, particularly in relation to signalling specificity between these two receptors
• mechanisms regulating translocation and activity of glucose transporters
• cellular mechanisms of insulin resistance, particularly as induced by tumour necrosis factor alpha
• differentiation, metabolism and apoptosis of adipose cells

Specific techniques used

• generation of specific antibodies using peptide conjugates or GST-fusion proteins as immunogens
• immunological approaches to studying protein structure, function and subcellular location
• mammalian cell culture including 3T3-L1 adipocytes
• subcellular fractionation

Specific reagents available

• antibodies for insulin receptors, IGF receptors, insulin receptor substrates and various signalling adaptors
• chimeric TrkC-IR and TrkC-IGFR cDNA constructs
• epitope-tagged constructs for various insulin receptor substrates and adaptor proteins

Peter Shepherd

Research areas

• role of PI-3-kinase in insulin signal transduction
• role of the mammalian target of rapamycin in insulin signalling
• insulin signalling and insulin resistance in muscle including cultured models of human muscle
• molecular mechanisms of cholesterol ester accumulation in foam cells in diabetes and role in cardiovascular disease

Specific reagents available

• reagents related to PI-3-kinase are available