Flow Cytometric Quantification of Glucose-Stimulated β-Cell Metabolic Flux Can Reveal Impaired Islet Functional Potency

Cell Transplantation

Article

Matthew S. Hanson¹  Luis A. Fernandez¹  Juan S. Danobeitia¹  Anja Steffen²  Barbara Ludwig² 

[1] Department of Surgery, Division of Transplantation, University of Wisconsin-Madison, Madison, WI, USA;Medizinische Klinik und Poliklinik III, University of Carl Gustav Carus, Dresden, Germany;

关键词: Islet;    β-Cell;    Glucose;    Mitochondria;    Transplantation;    Diabetes;    Pancreas;    Flow cytometry;   

DOI  :  10.3727/096368908787648038

 received in 2008-02-22, accepted in 2008-07-09,  发布年份 2008

来源: Sage Journals

【 摘 要 】

The objective of this study was to develop a multiparametric flow cytometry assay to simultaneously quantify isolated pancreatic islet cell viability, apoptosis, and glucose-induced metabolic flux. INS-1 and rat islet β-cells were stained with fluorescent probes for cell viability (ToPro3), apoptosis (Annexin V and VADFMK), and intracellular calcium (Ca2+i) (Fura Red), stimulated with glucose, and analyzed on a FACS VantageTM flow cytometer. Glucose-induced metabolic activity was indicated by changes in Fura Red fluorescence and the autofluorescence of the pyridine [NAD(P)H] and flavin (FAD/FMN) nucleotides. Rat islets cultured under conditions of proinflammatory cytokine-induced oxidative stress were evaluated by flow cytometry and transplantation into diabetic mice. INS-1 and rat islet β-cell health and metabolic activity were quantified in response to elevated glucose dose and inhibitors of glycolysis and mitochondrial function. Changes in metabolite fluorescence were converted to an area under the curve (AUC) value. Rat islets cultured under oxidative stress conditions showed decreased viability, increased apoptosis, and decreased glucose-induced metabolic activity indicated by reduced AUC for pyridine and flavin nucleotides and Ca2+i. Reduced metabolite AUC measured by flow cytometry correlated with the inability to reverse diabetes in mice. Single cell flow cytometry can simultaneously quantify both overall islet cell health and β-cell glucose responsiveness as indicators of functional potency.

【 授权许可】

Unknown   
© 2008 Cognizant Comm. Corp.