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.

Figure 1

JC-1 localization in representative confocal images of mononuclear cells from a subject with T2DM (a,b,c) and a subject without diabetes (d,e,f). (a,d) Localization of red JC-1 fluorescent intensity following excitation with a wavelength of 488 nm. (b,e) Green JC-1 fluorescence intensity following excitation with the same wavelength. (c,f) Overlay for green and red JC-1 fluorescence, showing co-localization to the same area of the cells. The red:green fluorescence ratio for the diabetic and non-diabetic cells were 1.88 and 1.19, respectively.

Figure 2

Representative flourscent confocal images of human mononuclear cells with NAO (a,b) and MitoSox™ (c,d) fluorophores for a patient with T2DM (a,c) and without diabetes (b,d

Figure 3

Mitochondrial membrane potential as estimated by JC-1 is appropriately depolarized by FCCP exposure. Mononuclear cells were incubated for 30 minutes in the indicated concentrations of FCCP.

Figure 4

Differences in mitochondrial morphology between patients with T2DM and controls. Displayed are electron microscopy images (original magnification 12,000×, print magnification 23900× @8 in, 500 nm scale) showing that the mitochondria of Type 2 diabetics (a) are generally smaller and rounder compared to non diabetic controls (b). White arrows point to representative mitochondria in each image.

Figure 5

The mitochondria patients with T2DM are more highly polarized (more negative) than controls subjects as measured by the red:green fluorescent intensity ration of theJC-1 probe. Data presented as mean±S.E. *− P=0.02

Figure 6

Mitochondrial mass/cardiolipin content is lower in patients with T2DM relative to control subjects as measured by nonyl acridine orange fluorescent intensity *−P=0.008

Figure 7

Mitochondrial superoxide production is higher in a subset patients with T2DM relative to control subjects as measured by MitoSox™. Data presented as mean±S.E. *−P=0.01