Trans-plasma membrane electron transport in muscle cells. Shannon Kelly

Transcription

1 Trans-plasma membrane electron transport in muscle cells Shannon Kelly

2 Introduction Trans-plasma membrane electron transport (tpmet) Shuttle-based electron transfer Enzyme-mediated electron transfer Intracellular DHA Ascorbate NADP+ Oxidase Extracellular DHA Ascorbate O 2 O 2.

3 Introduction Cellular Functions Cell growth (Crane et al. 1985, Sun et al 1987) Cell viability (Larm et al 1994) Iron metabolism (Inman et al 1994) Cell signaling (del Castillo-Olivares et al 1995, Medina et al 1992) Protection against reactive oxygen species (Diaz-Gomez et al 1997). Contribute to the development of disease Cancer (Herst and Berridge 2007) Cardiovascular disease (Baoutina et al 2001) Metabolic syndrome (Furukawa et al 2004)

4 Research Aims To elucidate the mechanism of tpmet by C2C12 myotubes Specific Aim 1: To determine the role of superoxide in tpmet Specific Aim 2: To determine the role of glycolysis and the pentose phosphate pathway in tpmet Specific Aim 3: To determine if enzymes involved in the mechanism are co-localized

5 Methods Intracellular Extracellular C2C12 myotubes Immortal mouse muscle cell line Water soluble tetrazolium salt-1 (WST-1) Extracellular electron acceptor ELECTRONS PMS WST-1 Formazan

6 Addition of superoxide dismutase (SOD) suppresses tpmet 140 nmol WST-1 reduction per µg protein (-) SOD (+) SOD Time (minutes) Kelly et al. Submitted

7 Intracellular Extracellular O 2 O 2.

8 Addition of GKT suppresses tpmet nmol WST-1 reduction per µg protein at 60 minutes p<0.05 vs 0uM p<0.05 vs 5uM p<0.05 vs 10uM GKT concentration (µm)

9 Intracellular Extracellular NADP+ O 2 OXIDASE. O 2

10 tpmet is a glucose dependent process 120 nmol WST-1 reduction per µg protein at 60 minutes Glucose concentration (mm) nmol WST-1 reduction per µg protein at 60 min Glucose concentration (mm)

11 Glucose can rescue the suppression of tpmet 70 nmol WST-1 reduction per µg protein at 60 min Glucose Pyruvate

12 Intracellular Extracellular TRANSPORTER NADP+ O 2 OXIDASE. O 2

13 2-deoxy-D-glucose suppresses tpmet nmol WST-1 reduction per µg protein Time (minutes) Pyruvate Glucose 2DG

14 Intracellular Extracellular -6- PHOSPHATE HEXOKINASE TRANSPORTER NADP+ O 2 OXIDASE. O 2

15 Addition of dehydroepiandrosterone (DHEA) suppresses tpmet 70 nmol WST-1reduction per µg protein Time (minutes) (-) DHEA (+) DHEA

16 Intracellular Extracellular -6- PHOSPHATE HEXOKINASE TRANSPORTER -6- PHOSPHATE DEHYDROGENASE NADP+ NADP+ O 2 OXIDASE. O 2 6-PHOSPHOGLUCONO-d -LACTONE

17 Glycolysis inhibitors do not suppress tpmet 80 nmol WST-1 reduction per µg of protein at 60 minutes (-) Inhibitor (+) Inhibitor 0 3PO NaF Laboratoryinfo.com

18 Intracellular Extracellular GLYCOLYSIS -6- PHOSPHATE HEXOKINASE TRANSPORTER -6- PHOSPHATE DEHYDROGENASE NADP+ NADP+ O 2 OXIDASE. O 2 6-PHOSPHOGLUCONO-d -LACTONE GROWTH AND METABOLISM INSULIN RECEPTOR

19 Increase in tpmet does not activate insulin signaling 1.2 pakt AKT Glucose concentration (mm) pakt/akt (relative to control) mm 0 mm Glucose concentration

20 Intracellular Extracellular GLYCOLYSIS -6- PHOSPHATE HEXOKINASE TRANSPORTER -6- PHOSPHATE DEHYDROGENASE NADP+ NADP+ O 2 OXIDASE. O 2 6-PHOSPHOGLUCONO-d -LACTONE GROWTH AND METABOLISM INSULIN RECEPTOR

21 Future directions Determine the specific oxidase (NOX) involved in the metabolon Determine the co-localization of the enzymes in the metabolon

22 Acknowledgements Dr. Fisher Committee Members Dr. Blythe Janowiak Dr. Brain Downes Fisher Lab Amanda Eccardt Mark Mannino Neej Patel Lyn Mattathil Thomas Bell Dr. Koyal Garg

23 References Crane, F. L., Sun, I. L., Clark, M. G., Grenbing, C. & Low, H. Transplasma-membrane redox systems in growth and development. Biochim Biophys Acta (1985). Sun, I. L., Navas, P., Crane, F. L., Morre, D. J. & Low, H. NADH diferric transferrin reductase in liver plasma membrane. J Biol Chem. 262 (33), (1987). Larm, J. A., Vaillant, F., Linnane, A. W. & Lawen, A. Up-regulation of the plasma membrane oxidoreductase as a prerequisite for the viability of human Namalwa rho 0 cells. J Biol Chem. 269 (48), (1994). Inman, R. S., Coughlan, M. M. & Wessling-Resnik, M. Extracellular ferrireductase activity of K562 cells is coupled to transferrin-independent iron transport. Biochemistry (1994). del Castillo-Olivares, A., Esteban del Valle, A., Marquez, J., Nunez de Castro, I. & Medina, M. A. Ehrlich cell plasma membrane redox system is modulated through signal transduction pathways involving cgmp and Ca2+ as second messengers. J Bioenerg Biomembr. 27 (6), (1995). Medina, M. A., del Castillo-Olivares, A. & Schweigerer, L. Plasma membrane redox activity correlates with N-myc expression in neuroblastoma cells. FEBS Lett. 311 (2), (1992). Diaz-Gomez, C., Villalba, J. M., Perez-Vicente, R. & Crane, F. Ascorbate Stabilization Is Stimulated in rho(0)hl-60 Cellsby CoQ10 Increase at the Plasma Membrane. Biochem Biophys Res Commun. 234 (1), (1997). Herst, P. M. & Berridge, M. V. Cell surface oxygen consumption: a major contributor to cellular oxygen consumption in glycolytic cancer cell lines. Biochim Biophys Acta (2), , doi: /j.bbabio , (2007). Baoutina, A., Dean, R. T. & Jessup, W. Trans-plasma membrane electron transport induces macrophage-mediated low density lipoprotein oxidation. FASEB J. 15 (9), (2001). Furukawa, S. et al. Increased oxidative stress in obesity and its impact on metabolic syndrome. J Clin Invest. 114 (12), , doi: /jci21625, (2004).

24 Addition of GSK activates tpmet nmol WST-1 reduction per µg protein at 60 minutes p<0.05 vs 0 µm GSK concentration (µm)

25 Oxidase Activity Assays Brain Heart Kidney Liver TA C2C12 L6 SOL EDL Brain Heart Kidney Liver TA C2C12 L6 SOL EDL -GKT +GKT -GSK +GSK L6 = rat muscle cell line TA = Tibialis anterior SOL = Soleus EDL = Extensor digitorum longus

26 Oxidase Western Blots Brain Heart Kidney Liver TA C2C12 L6 SOL EDL Brain Heart Kidney Liver TA C2C12 L6 SOL EDL NOX 1 NOX 2 NOX 4 GAPDH GAPDH