title: Comparative Analysis of the Mitochondrial Physiology of Pancreatic β Cells.
abstract: The mitochondrial metabolism of β cells is thought to be highly specialized. Its direct comparison with other cells using isolated mitochondria is limited by the availability of islets/β cells in sufficient quantity. In this study, we have compared mitochondrial metabolism of INS1E/β cells with other cells in intact and permeabilized states. To selectively permeabilize the plasma membrane, we have evaluated the use of perfringolysin-O (PFO) in conjunction with microplate-based respirometry. PFO is a protein that binds membranes based on a threshold level of active cholesterol. Therefore, unless active cholesterol reaches a threshold level in mitochondria, they are expected to remain untouched by PFO. Cytochrome c sensitivity tests showed that in PFO-permeabilized cells, the mitochondrial integrity was completely preserved. Our data show that a time-dependent decline of the oligomycin-insensitive respiration observed in INS1E cells was due to a limitation in substrate supply to the respiratory chain. We predict that it is linked with the β cell-specific metabolism involving metabolites shuttling between the cytoplasm and mitochondria. In permeabilized β cells, the Complex l-dependent respiration was either transient or absent because of the inefficient TCA cycle. The TCA cycle insufficiency was confirmed by analysis of the CO2evolution. This may be linked with lower levels of NAD+, which is required as a co-factor for CO2producing reactions of the TCA cycle. β cells showed comparable OxPhos and respiratory capacities that were not affected by the inorganic phosphate (Pi) levels in the respiration medium. They showed lower ADP-stimulation of the respiration on different substrates. We believe that this study will significantly enhance our understanding of the β cell mitochondrial metabolism.
authors.affiliation: Pioneer Valley Life Sciences Institute, Springfield, MA, USA.
authors.forename: Chul
authors.initials: C
authors.lastname: Kim
authors.affiliation: Pioneer Valley Life Sciences Institute, Springfield, MA, USA.
authors.forename: Pinal
authors.initials: P
authors.lastname: Patel
authors.affiliation: Departments of Biochemistry and Molecular Biology, University of Massachusetts, Amherst, MA, USA.
authors.forename: Lindsey M
authors.initials: LM
authors.lastname: Gouvin
authors.affiliation: Pioneer Valley Life Sciences Institute, Springfield, MA, USA.
authors.forename: Melissa L
authors.initials: ML
authors.lastname: Brown
authors.affiliation: Department of Biology, University of Massachusetts, Amherst, MA, USA.
authors.forename: Ahmed
authors.initials: A
authors.lastname: Khalil
authors.affiliation: Pioneer Valley Life Sciences Institute, Springfield, MA, USA.
authors.forename: Elizabeth M
authors.initials: EM
authors.lastname: Henchey
authors.affiliation: Departments of Biochemistry and Molecular Biology, University of Massachusetts, Amherst, MA, USA.
authors.forename: Alejandro P
authors.initials: AP
authors.lastname: Heuck
authors.affiliation: Pioneer Valley Life Sciences Institute, Springfield, MA, USA ; Department of Biology, University of Massachusetts, Amherst, MA, USA ; Division of Endocrinology, Diabetes & Metabolism at Baystate Medical Center of Tufts University School of Medicine, Springfield, MA, USA.
authors.forename: Nagendra
authors.initials: N
authors.lastname: Yadava
keywords: Mitochondrial metabolism; OxPhos; Oxidative phosphorylation; Perfringolysin-O; Respiratory chain; Respirometry