title: Measuring mitochondrial uncoupling protein-2 level and activity in insulinoma cells.
abstract: Mitochondrial uncoupling protein-2 (UCP2) regulates glucose-stimulated insulin secretion (GSIS) by pancreatic beta cells-the physiological role of the beta cell UCP2 remains a subject of debate. Experimental studies informing this debate benefit from reliable measurements of UCP2 protein level and activity. In this chapter, we describe how UCP2 protein can be detected in INS-1 insulinoma cells and how it can be knocked down by RNA interference. We demonstrate briefly that UCP2 knockdown lowers glucose-induced rises in mitochondrial respiratory activity, coupling efficiency of oxidative phosphorylation, levels of mitochondrial reactive oxygen species, and insulin secretion. We provide protocols for the detection of the respective UCP2 phenotypes, which are indirect, but invaluable measures of UCP2 activity. We also introduce a convenient method to normalize cellular respiration to cell density allowing measurement of UCP2 effects on specific mitochondrial oxygen consumption.
authors.affiliation: School of Biomedical and Biological Sciences, Plymouth University, Drake Circus, Plymouth, United Kingdom.
authors.forename: Jonathan
authors.initials: J
authors.lastname: Barlow
authors.forename: Verena
authors.initials: V
authors.lastname: Hirschberg
authors.forename: Martin D
authors.initials: MD
authors.lastname: Brand
authors.forename: Charles
authors.initials: C
authors.lastname: Affourtit
mesh_terms: D000818:Animals; D015153:Blotting, Western; D002452:Cell Count; D045744:Cell Line, Tumor; D019069:Cell Respiration; D015870:Gene Expression; D005947:Glucose / Q000378:metabolism / Q000494:pharmacology; D007328:Insulin / Q000096:biosynthesis / Q000378:metabolism; D000078790:Insulin Secretion; D007340:Insulinoma / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D007473:Ion Channels / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D008928:Mitochondria / Q000378:metabolism*; D024101:Mitochondrial Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D010085:Oxidative Phosphorylation; D010190:Pancreatic Neoplasms / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D051381:Rats; D017382:Reactive Oxygen Species / Q000378:metabolism*; D000071246:Uncoupling Protein 2
chemical_list: D007328:Insulin; D007473:Ion Channels; D024101:Mitochondrial Proteins; D034741:RNA, Small Interfering; D017382:Reactive Oxygen Species; C000604391:Ucp2 protein, rat; D000071246:Uncoupling Protein 2; D005947:Glucose
keywords: Cellular bioenergetics; Coupling efficiency of oxidative phosphorylation; Glucose-stimulated insulin secretion; Insulinoma cells; Mitochondrial uncoupling protein-2 (UCP2); Pancreatic beta cells; Reactive oxygen species; Type 2 diabetes; UCP2 activity; UCP2 detection and knockdown