title: A novel high-throughput assay for islet respiration reveals uncoupling of rodent and human islets.
abstract: BACKGROUND
  The pancreatic beta cell is unique in its response to nutrient by increased fuel oxidation. Recent studies have demonstrated that oxygen consumption rate (OCR) may be a valuable predictor of islet quality and long term nutrient responsiveness. To date, high-throughput and user-friendly assays for islet respiration are lacking. The aim of this study was to develop such an assay and to examine bioenergetic efficiency of rodent and human islets.
  
  
  RESULTS
  The XF24 respirometer platform was adapted to islets by the development of a 24-well plate specifically designed to confine islets. The islet plate generated data with low inter-well variability and enabled stable measurement of oxygen consumption for hours. The F1F0 ATP synthase blocker oligomycin was used to assess uncoupling while rotenone together with myxothiazol/antimycin was used to measure the level of non-mitochondrial respiration. The use of oligomycin in islets was validated by reversing its effect in the presence of the uncoupler FCCP. Respiratory leak averaged to 59% and 49% of basal OCR in islets from C57Bl6/J and FVB/N mice, respectively. In comparison, respiratory leak of INS-1 cells and C2C12 myotubes was measured to 38% and 23% respectively. Islets from a cohort of human donors showed a respiratory leak of 38%, significantly lower than mouse islets.
  
  
  CONCLUSIONS
  The assay for islet respiration presented here provides a novel tool that can be used to study islet mitochondrial function in a relatively high-throughput manner. The data obtained in this study shows that rodent islets are less bioenergetically efficient than human islets as well as INS1 cells.
authors.affiliation: Department of Medicine, Boston University School of Medicine, Boston, Massachusetts, United States of America.
authors.forename: Jakob D
authors.initials: JD
authors.lastname: Wikstrom
authors.forename: Samuel B
authors.initials: SB
authors.lastname: Sereda
authors.forename: Linsey
authors.initials: L
authors.lastname: Stiles
authors.forename: Alvaro
authors.initials: A
authors.lastname: Elorza
authors.forename: Emma M
authors.initials: EM
authors.lastname: Allister
authors.forename: Andy
authors.initials: A
authors.lastname: Neilson
authors.forename: David A
authors.initials: DA
authors.lastname: Ferrick
authors.forename: Michael B
authors.initials: MB
authors.lastname: Wheeler
authors.forename: Orian S
authors.initials: OS
authors.lastname: Shirihai
mesh_terms: D000328:Adult; D000818:Animals; D002259:Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone / Q000494:pharmacology; D002460:Cell Line; D019069:Cell Respiration / Q000187:drug effects / Q000502:physiology; D003924:Diabetes Mellitus, Type 2 / Q000378:metabolism; D005260:Female; D057166:High-Throughput Screening Assays / Q000379:methods; D006801:Humans; D066298:In Vitro Techniques; D050417:Insulin-Secreting Cells / Q000187:drug effects / Q000378:metabolism; D007515:Islets of Langerhans / Q000187:drug effects / Q000378:metabolism*; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D008875:Middle Aged; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism; D009840:Oligomycins / Q000494:pharmacology; D010101:Oxygen Consumption / Q000187:drug effects / Q000502:physiology; D013045:Species Specificity; D014475:Uncoupling Agents / Q000494:pharmacology; D055815:Young Adult
chemical_list: D009840:Oligomycins; D014475:Uncoupling Agents; D002259:Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone