title: Altered mitochondrial membrane potential, mass, and morphology in the mononuclear cells of humans with type 2 diabetes.
abstract: Mitochondrial membrane hyperpolarization and morphologic changes are important in inflammatory cell activation. Despite the pathophysiologic relevance, no valid and reproducible method for measuring mitochondrial homeostasis in human inflammatory cells is available currently. The purpose of this study was to define and validate reproducible methods for measuring relevant mitochondrial perturbations and to determine whether these methods could discern mitochondrial perturbations in type 2 diabetes mellitus (T2DM), which is a condition associated with altered mitochondrial homeostasis. We employed 5,5',6,6'-tetrachloro-1,1'3,3'-tetraethylbenzamidazol-carboncyanine (JC-1) to estimate mitochondrial membrane potential (Psi(m)) and acridine orange 10-nonyl bromide (NAO) to assess mitochondrial mass in human mononuclear cells isolated from blood. Both assays were reproducible. We validated our findings by electron microscopy and pharmacologic manipulation of Psi(m). We measured JC-1 and NAO fluorescence in the mononuclear cells of 27 T2DM patients and 32 controls. Mitochondria were more polarized (P = 0.02) and mitochondrial mass was lower in T2DM (P = 0.008). Electron microscopy demonstrated diabetic mitochondria were smaller, were more spherical, and occupied less cellular area in T2DM. Mitochondrial superoxide production was higher in T2DM (P = 0.01). Valid and reproducible measurements of mitochondrial homeostasis can be made in human mononuclear cells using these fluorophores. Furthermore, potentially clinically relevant perturbations in mitochondrial homeostasis in T2DM human mononuclear cells can be detected.
authors.affiliation: Department of Medicine, Division of Cardiovascular Medicine, Medical College of Wisconsin, Milwaukee, WI 53226, USA. mwidlans@mcw.edu
authors.forename: Michael E
authors.initials: ME
authors.lastname: Widlansky
authors.forename: Jingli
authors.initials: J
authors.lastname: Wang
authors.forename: Sherene M
authors.initials: SM
authors.lastname: Shenouda
authors.forename: Tory M
authors.initials: TM
authors.lastname: Hagen
authors.forename: Anthony R
authors.initials: AR
authors.lastname: Smith
authors.forename: Tinoy J
authors.initials: TJ
authors.lastname: Kizhakekuttu
authors.forename: Matthew A
authors.initials: MA
authors.lastname: Kluge
authors.forename: Dorothee
authors.initials: D
authors.lastname: Weihrauch
authors.forename: David D
authors.initials: DD
authors.lastname: Gutterman
authors.forename: Joseph A
authors.initials: JA
authors.lastname: Vita
mesh_terms: D000328:Adult; D000368:Aged; D000609:Aminoacridines / Q000378:metabolism; D001562:Benzimidazoles / Q000378:metabolism; D015415:Biomarkers; D002232:Carbocyanines / Q000378:metabolism; D002308:Cardiolipins / Q000378:metabolism; D016022:Case-Control Studies; D003430:Cross-Sectional Studies; D003924:Diabetes Mellitus, Type 2 / Q000378:metabolism*; D005260:Female; D005456:Fluorescent Dyes / Q000378:metabolism; D006801:Humans; D008214:Lymphocytes / Q000166:cytology / Q000378:metabolism* / Q000648:ultrastructure; D008297:Male; D053078:Membrane Potential, Mitochondrial / Q000502:physiology*; D008875:Middle Aged; D008928:Mitochondria / Q000378:metabolism / Q000648:ultrastructure*; D009000:Monocytes / Q000166:cytology / Q000378:metabolism* / Q000648:ultrastructure; D015203:Reproducibility of Results; D013481:Superoxides / Q000378:metabolism
chemical_list: D000609:Aminoacridines; D001562:Benzimidazoles; D015415:Biomarkers; D002232:Carbocyanines; D002308:Cardiolipins; D005456:Fluorescent Dyes; D013481:Superoxides; C068624:5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolocarbocyanine; C041691:10-N-nonylacridinium orange