Laboratory of Cellular Metabolism

Head: Krzysztof ZABŁOCKI

Staff: Joanna Bandorowicz-Pikuła, Beata Drabarek (PhD student), Dorota Dymkowska, Justyna Jakubczyk, Wanda Kłopocka, Elżbieta Krasowska (PhD student), Marta Onopiuk, Marcin Woś (PhD student)

Research profile:

Our research is focused on energy metabolism, intracellular calcium handling and signal transduction in relation to cellular metabolism in normal and pathological conditions. Specifically, we are interested in:

• calcium homeostasis, regulation of calcium signals and changes in energy metabolism in muscle cells derived from dystrophic mouse (mdx) in addition, we investigate dystrophy-related changes in calcium homeostasis in neurons
• diabetes-related alterations of energy metabolism of muscle, liver, and vascular endothelium
• mitochondria as a potential pharmaceutical target in insulin resistance therapy and prevention of type 2 diabetes
• human metabolic diseases related to abnormal transport and storage of cholesterol in the cell with special emphasis on signal transduction pathways via plasma membrane microdomain
• the role of calcium response and αvβ5 integrin activation in P2Y2 receptor stimulation and cell recovery from ROCK inhibition

Methods:

• cell culture
• western blotting
• measurement of cellular respiration
• spectrofluorimetry
• confocal microscopy techniques
• flow cytometry
• laser scanning cytometry
• duolink – “In-cell Co-IP”
• real-time PCR
HPLC
• siRNA
• cell transfection to overexpress specific genes

Current research activities:

• Investigating dystrophy-related alterations of nucleotide receptors (mainly various P2Y and P2X7) and store-operated calcium channels in myoblasts and neurons derived from dystrophic mdx mouse. Myoblast are undifferentiated muscle cells, thus expression of the dystrophin encoding gene is not observed therein. Despite this, numerous phenotypic changes particularly concerning intracellular calcium homeostasis and mitochondrial energy metabolism have been identified in our laboratory. However, the molecular mechanism responsible for this phenomenon is still elusive. Recently we have focused our attention on differences in microRNA content in normal and dystrophic cells. We are also interested in changes of calcium homeo- stasis in neurons of dystrophic mouse
• Prevention of insulin resistance and hyperglycemia- induced pathological changes in myotubes, hepatocytes, vascular endothelium based on the regulation of PPAR receptor activity and modulation of mitochondrial metabolism using innovative agonists. Explanation of mitochondria-related effects of hyperglycemia (with spe- cial attention to reactive oxygen species formation) in endothelial cells
• Characterization of cholesterol and sphingomyelin- enriched microdomains in fibroblasts from Niemann-Pick type C patients: identification of signal transduction path- ways related to the abnormal storage of cholesterol in these cells
• Stimulation of Rac1-mediated pathway via Go and P2Y2 receptor is possible only when direct interaction between receptor and αvβ5 integrins occurs. In glioma C6 cells this interaction is blocked by RGD and by antibody against αvβ5 integrins (PIF76) as shown by immunoprecipitation. Similarly, it is also inhibited when cells are stimulated in calcium-free medium. For Go–mediated Rac1 activation and stimulation Rac1/PAK/LIMK signaling pathway calcium influx from extracellular space is necessary

Selected publications:

Kłopocka W., Korczyński J., Pomorski P. (2013) Cytoskel- eton and nucleotide signaling in Glioma C6 Cells. Glioma Signaling, Advances in Experimental Medicine and Biology, 986: 103-119.

Drabarek B., Dymkowska D., Szczepanowska J., Zabłocki K. (2012) “TNFalpha affects energy metabolism and stimulates biogenesis of mitochondria in EA.hy926 endothelial cells”. The International Journal of Biochemistry & Cell Biology, 44: 1390-1397.

Sztolsztener M.E., Dobrzyń A., Pikuła S., Tylki-Szymańska A., Bandorowicz-Pikuła J. (2012) Impaired dynamics of late endosome/lysosome compartment in human Niemann- Pick type C skin fibroblasts carrying mutation in NPC1 gene. Molecular BioSystems, 8: 1197-1205.

Young C., Brutkowski W., Lien C-F., Arkle S., Lochmüller H., Zabłocki K., Górecki, D.C. (2012) P2×7 purinoceptor altera- tions in dystrophic mdx mouse muscles: Relationship to pathology and potential target for treatment. Journal of Cellular and Molecular Medicine, 16: 1026-1037.

Onopiuk M., Brutkowski W., Wierzbicka K., Wojciechowska S., Szczepanowska J., Fronk J., Lochmüller H., Górecki D.C., Zabłocki K. (2009) Mutation in dystrophin-encoding gene affects energy metabolism in mouse myoblasts. Biochemical and Biophysical Research Communications, 386: 463-466