Flow cytometric quantification of glucose-stimulated beta-cell metabolic flux can reveal impaired islet functional potency.
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 beta-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 Vantage 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 beta-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 beta-cell glucose responsiveness as indicators of functional potency.
Department of Surgery, Division of Transplantation, University of Wisconsin-Madison, Madison, WI 53792-3236, USA.
Matthew S
MS
Hanson
Anja
A
Steffen
Juan S
JS
Danobeitia
Barbara
B
Ludwig
Luis A
LA
Fernandez
D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D002118:Calcium / Q000378:metabolism; D002470:Cell Survival / Q000187:drug effects; D005486:Flavin Mononucleotide / Q000378:metabolism; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D005434:Flow Cytometry / Q000379:methods; D005947:Glucose / Q000494:pharmacology*; D006801:Humans; D007328:Insulin / Q000378:metabolism; D000078790:Insulin Secretion; D050417:Insulin-Secreting Cells / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D007515:Islets of Langerhans / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D016381:Islets of Langerhans Transplantation / Q000009:adverse effects / Q000502:physiology*; D008297:Male; D008356:Mannoheptulose / Q000494:pharmacology; D009243:NAD / Q000378:metabolism; D009249:NADP / Q000378:metabolism; D018384:Oxidative Stress / Q000187:drug effects; D051381:Rats; D011917:Rats, Inbred Lew; D012402:Rotenone / Q000494:pharmacology
D007328:Insulin; D012402:Rotenone; D009243:NAD; D005182:Flavin-Adenine Dinucleotide; D009249:NADP; D008356:Mannoheptulose; D005486:Flavin Mononucleotide; D005947:Glucose; D002118:Calcium