title: A new mitochondrial pH biosensor for quantitative assessment of pancreatic β-cell function.
abstract: Mitochondrial pH is a key determinant of mitochondrial energy metabolism. We have developed a new fluorescence-based ratiometric pH biosensor using a chloride-insensitive and hydrogen-sensitive probe for direct, quantitative and bleaching-free measurement in a living cell. Fusing this biosensor with a mitochondrial localization signal (MTpHGV) allowed us to determine mitochondrial pH. This new system was applied to measure mitochondrial pH in pancreatic β-cells, in which mitochondrial function plays a pivotal role in insulin secretion. Rat INS1 cells and mouse MIN6 cells are transfected with MTpHGV stably to monitor mitochondrial pH. While carbonyl cyanide 3-chlorophenylhydrazone (CCCP) treatment rapidly decreased mitochondrial pH in cultured rat MTpHGV-INS-1 cells, MTpHGV-MIN6 cells showed a rapid increase. These data suggest that MTpHGV probe exist in matrix side in INS-1 cells, but on the outer side of mitochondrial inner membrane in MIN6 cells. Moreover, while MTpHGV-INS-1 cells showed a rapid increase of pH by glucose stimulation, mitochondrial pH decreased quickly by glucose stimulation in all MTpHGV-MIN6 cells examined and recovered smoothly. Perfusion study of glucose load in MTpHGV-MIN6 cells under aminooxyacetate (AOA) or 100μM diazoxide showed that this mitochondrial pH acidification was dependent on nicotinamide adenine dinucleotide (NADH) shuttle, but independent from KATP channel. This new system for measuring mitochondrial pH is sensitive across the range of physiologic conditions and may be a useful tool for evaluating mitochondrial function in living cells.
authors.affiliation: Diabetes Center, Tokyo Women's Medical University, Tokyo, Japan. mogatamd@dmc.twmu.ac.jp
authors.forename: Makiko
authors.initials: M
authors.lastname: Ogata
authors.forename: Takeo
authors.initials: T
authors.lastname: Awaji
authors.forename: Naoko
authors.initials: N
authors.lastname: Iwasaki
authors.forename: Risa
authors.initials: R
authors.lastname: Fujimaki
authors.forename: Miho
authors.initials: M
authors.lastname: Takizawa
authors.forename: Kei
authors.initials: K
authors.lastname: Maruyama
authors.forename: Yasuhiko
authors.initials: Y
authors.lastname: Iwamoto
authors.forename: Yasuko
authors.initials: Y
authors.lastname: Uchigata
mesh_terms: D000625:Aminooxyacetic Acid / Q000494:pharmacology; D000818:Animals; D015374:Biosensing Techniques; D002118:Calcium / Q000378:metabolism; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone / Q000494:pharmacology; D002460:Cell Line; D003981:Diazoxide / Q000494:pharmacology; D005947:Glucose / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007328:Insulin / Q000378:metabolism; D000078790:Insulin Secretion; D050417:Insulin-Secreting Cells / Q000737:chemistry* / Q000187:drug effects / Q000378:metabolism; D051379:Mice; D008928:Mitochondria / Q000737:chemistry*; D061209:Proton Ionophores / Q000494:pharmacology; D051381:Rats
chemical_list: D007328:Insulin; D061209:Proton Ionophores; D000625:Aminooxyacetic Acid; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone; D005947:Glucose; D003981:Diazoxide; D002118:Calcium