Ardenkjær-Larsen, Jan Henrik

Ghiasi, Seyed M.

Jensen, Pernille Rose

Malinowski, Ronja Maja

Mandrup-Poulsen, Thomas

Online Research Database In Technology

                       General rights Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights.  • Users may download and print one copy of any publication from the public portal for the purpose of private study or research. • You may not further distribute the material or use it for any profit-making activity or commercial gain • You may freely distribute the URL identifying the publication in the public portal   If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim.     Downloaded from orbit.dtu.dk on: Apr 10, 2018Probing of biochemical pathways in clonal pancreatic –cells by quantitative dDNP ofmetabolite extractsMalinowski, Ronja Maja; Ghiasi, Seyed M.; Mandrup-Poulsen, Thomas; Jensen, Pernille Rose;Ardenkjær-Larsen, Jan HenrikPublication date:2017Document VersionPublisher's PDF, also known as Version of recordLink back to DTU OrbitCitation (APA):Malinowski, R. M., Ghiasi, S. M., Mandrup-Poulsen, T., Jensen, P. R., & Ardenkjær-Larsen, J. H. (2017).Probing of biochemical pathways in clonal pancreatic –cells by quantitative dDNP of metabolite extracts.Abstract from EUROMAR 2017, Warsaw, Poland.Probing of biochemical pathways in clonal pancreatic β–cells by quantitative dDNP of metabolite extracts   Ronja M. Malinowski1, Seyed M. Ghiasi2, Thomas Mandrup-Poulsen2, Pernille R. Jensen1, Jan H. Ardenkjær-Larsen1,3  1Technical University of Denmark, Kgs. Lyngby, Denmark, 2University of Copenhagen, Copenhagen, Denmark, 3GE Healthcare, Denmark        Anaplerotic flux into the tricarboxylic (TCA) cycle is crucial for glucose-stimulated insulin secretion (GSIS) in pancreatic β-cells [1]. Although it is well-established that GSIS requires oxidation of carbohydrates in the mitochondria to drive ATP production, studies have suggested intermediates generated directly or indirectly from anaplerosis may act as second messengers linking glucose oxidation to insulin release [2].      In this study, dissolution Dynamic Nuclear Polarization (dDNP) is used to visualize the metabolic fate of [13C6, D6]glucose (13C-Glc) in cell extracts from the clonal pancreatic cell-line INS-1 from rat at two different 13C-Glc concentrations (11.7 mM and 17 mM). The extracts were obtained by incubation of 10 mio. cells in Eppendorf tube for 2, 4 and 8 h, at which the reaction was stopped with perchloric acid and the metabolites were extracted, and the sample extracts were hyperpolarized. The metabolites were quantified according to an internal standard (Fig. 1). Glc was linearly consumed at an average rate of 0.44 mM/h (initial concentration 11.7 mM) and 0.48 mM/h (initial concentration 17 mM) at the two incubation concentrations, while the output in terms of a sum of observable metabolites has increased by 66% from 11.7 mM to 17 mM 13C-Glc incubation. (p = 0.02).     In both cases, lactate increases linearly, with no significant difference (p > 0.05). However, pyruvate decreases over time, but is significantly (p = 0.02) increased at 17 mM vs 11.7 mM by 155% (sum of pyruvate for the two experiments). Therefore, the intersection when lac = pyr occurs later in 17 mM Glc, than 11.7 mM Glc. Glc derived pyruvate has been suggested to have a prominent role in GSIS as several pathways involving pyruvate has been described [3], and the upregulation is expected since GSIS correlates directly with Glc concentration and pyruvate cycling [4].                                                                                [1] Cline, G.W., LePinet, R.L., Papas K.K., Kibbey, R.G., Shulman, G.I., “13C NMR Isotopomer Analysis of Anaplerotic Pathways in INS-1 Cells”, J. Biol. Chem., 279 (43): 44370-44375, 2004 [2] MacDonal, M.J., Fahien, L.A., Brown, L.J., Hasan, N.M., Buss, J.D., Kendrick, M.A., “Perspective: emerging evidence for signa ling roles of mitochondrial anaplerotic products in insulin secretion”, Am. J. Physiol. Endocrinol. Metab., 288: E1-E15, 2005 [3] Jensen, M.V., Joseph, J.W., Ronnebaum, S.M., Burgess, S.C., Sherry, A.D., Newgard, C.B., “Metabolic cycling in control of glucose-stimulated insulin secretion”, Am. J. Physiol. Endocrinol. Metab., 295: E1287-E1297, 2008 [4] Lu, D., Muldet, H., Zhao, P., Burgess, S., Jensen, M., Kamzolova, S., Newgard, C.B., Sherry, A.D., “13C NMR isotopomer analysis reveals a connection between pyruvate cycling and glucose-stimulated insulin secretion (GSIS)”, PNAS, 99(5): 2708 - 2713   Fig. 1 - Metabolites generated from 13C Glucose 11.7 mM (left) and 17 mM (right) incubations of INS-1 cells at various time points. (Lac – lactate, Glu – glutamate, Ala – alanine, Pyr – pyruvate) 

Probing of biochemical pathways in clonal pancreatic β–cells by quantitative dDNP of metabolite extracts

http://orbit.dtu.dk/ws/files/142083675/EUROMAR2017RonjaMalinowski.pdf

Probing of biochemical pathways in clonal pancreatic β–cells by quantitative dDNP of metabolite extracts

Abstract

Similar works

Full text

Available Versions

Online Research Database In Technology