Altered mitochondrial membrane potential, mass, and morphology in the mononuclear cells of humans with type 2 diabetes

Abstract

Mitochondrial membrane hyperpolarization and morphological changes are important in inflammatory cell activation. Despite the pathophysiological relevance, no valid and reproducible method for measuring mitochondrial homeostasis in human inflammatory cells is currently available. This study's purpose 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), 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 (ψm) 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 pharmacological manipulation of ψm. 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, 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. Further, potential clinically relevant perturbations in mitochondrial homeostasis in T2DM human mononuclear cells can be detected.

Impairment of Mitochondrial–Nuclear Cross Talk in Neutrophils of Patients with Type 2 Diabetes Mellitus

“…Considerable research has demonstrated that free radical injury at the level of mitochondria severely affects neutrophil functional longevity [7]. Recently, we and others have delineated the role and clinical significance of mitochondrial dysfunction in peripheral blood lymphocytes of diabetic subjects [8,9]. In continuation to our previous investigation in the T2DM cohort, we speculate that perturbed interplay of mitochondria-nuclear cross talk may as well create an imbalance in oxidative function of neutrophils in T2DM subjects contributing to increased cellular demise.…”

Atractylenolide III Enhances Energy Metabolism by Increasing the SIRT-1 and PGC1α Expression with AMPK Phosphorylation in C2C12 Mouse Skeletal Muscle Cells

“…18) Therefore, we measured the effect of AIII on ATP levels and mitochondrial mass using an ATP assay and by staining with NAO, which binds to cardiolipin (a phospholipid specifically present on the mitochondrial membranes), 19) respectively. AIII dose-dependently increased myotube NAO fluorescence intensities (Fig.…”

Bioenergetics and the Oxidative Burst: Protocols for the Isolation and Evaluation of Human Leukocytes and Platelets

“…This is clinically important since mitochondrial dysfunction can initiate a series of cellular events that promote pro-inflammatory signaling pathways or lead to cell death. Several studies have characterized mitochondrial function of peripheral blood mononuclear cells and platelets in conditions such as fibromyalgia, diabetes, septic shock, and Alzheimer's disease [3][4][5][6][7] . For example, a recent study evaluated the bioenergetics of platelets as a marker for mitochondrial function and found that platelets from Type 2 diabetic patients had diminished mitochondrial oxygen consumption 7,8 .…”