Measuring mitochondrial bioenergetics in INS-1E insulinoma cells.
Pancreatic beta cells secrete insulin in response to raised blood glucose levels. This glucose-stimulated insulin secretion (GSIS) depends on mitochondrial function and is regulated by the efficiency with which oxidative metabolism is coupled to ATP synthesis. Uncoupling protein-2 (UCP2) affects this coupling efficiency and is therefore a plausible pathological and physiological regulator of GSIS. In this respect, it is important to be able to measure coupling efficiencies accurately. Here, we describe experimental protocols to determine the coupling efficiency of trypsinized INS-1E cells, a popular beta cell model, and we present practical details of our RNA interference studies to probe the effect of UCP2 knockdown on this efficiency. We also introduce a method to determine coupling efficiencies noninvasively in attached cells and discuss theoretical and practical aspects of a modular-kinetic approach to describe and understand cellular bioenergetics.
MRC Dunn Human Nutrition Unit, Cambridge, United Kingdom.
Charles
C
Affourtit
Martin D
MD
Brand
D000818:Animals; D045744:Cell Line, Tumor; D004734:Energy Metabolism; D055785:Gene Knockdown Techniques; D006801:Humans; D050417:Insulin-Secreting Cells / Q000737:chemistry / Q000378:metabolism; D007340:Insulinoma / Q000235:genetics / Q000378:metabolism*; D007473:Ion Channels / Q000032:analysis / Q000235:genetics* / Q000378:metabolism*; D007700:Kinetics; D008928:Mitochondria / Q000737:chemistry / Q000378:metabolism*; D024101:Mitochondrial Proteins / Q000032:analysis / Q000235:genetics* / Q000378:metabolism*; D010101:Oxygen Consumption; D034622:RNA Interference; D000071246:Uncoupling Protein 2
D007473:Ion Channels; D024101:Mitochondrial Proteins; C000604389:UCP2 protein, human; D000071246:Uncoupling Protein 2