Relation of mitochondrial oxygen consumption in peripheral blood mononuclear cells to vascular function in type 2 diabetes mellitus

Abstract

Recent studies have shown mitochondrial dysfunction and increased production of reactive oxygen species in peripheral blood mononuclear cells (PBMCs) and endothelial cells from patients with diabetes mellitus. Mitochondria oxygen consumption is coupled to adenosine triphosphate (ATP) production and also occurs in an uncoupled fashion during formation of reactive oxygen species by components of the electron transport chain and other enzymatic sites. We therefore hypothesized that diabetes would be associated with higher total and uncoupled oxygen consumption in PBMCs that would correlate with endothelial dysfunction. We developed a method to measure oxygen consumption in freshly isolated PBMCs and applied it to 26 patients with type 2 diabetes mellitus and 28 non-diabetic controls. Basal (192 ± 47 vs 161 ± 44 pmoles/min, p = 0.01), uncoupled (64 ± 16 vs 53 ± 13 pmoles/min, p = 0.007), and maximal (795 ± 87 vs 715 ± 128 pmoles/min, p=0.01) oxygen consumption rates were higher in diabetic patients compared to controls. There were no significant correlations between oxygen consumption rates and endothelium-dependent flow-mediated dilation measured by vascular ultrasound. Non-endothelium-dependent nitroglycerin-mediated dilation was lower in diabetics (10.1 ± 6.6 vs 15.8 ± 4.8%, p = 0.03) and correlated with maximal oxygen consumption (r = -0.64, p=0.001). In summary, we found that diabetes mellitus is associated with a pattern of mitochondrial oxygen consumption consistent with higher production of reactive oxygen species. The correlation between oxygen consumption and nitroglycerin-mediated dilation may suggest a link between mitochondrial dysfunction and vascular smooth muscle cell dysfunction that merits further study. Finally, the described method may have utility for the assessment of mitochondrial function in larger scale observational and interventional studies in humans.

Keywords: blood mononuclear cells; diabetes mellitus; mitochondria; oxygen consumption.

Figure 1

Oxygen consumption rates (OCR) in PBMC’s from control subjects (n=28) and patients with Type 2 diabetes mellitus (n=26). OCR was measured approximately every 8 minutes using an XF-96 analyzer as described in Methods. Basal and post-oligomycin (5 μmoles/L) rates were calculated by averaging the last four measurements after achieving a steady state. Sequential measurements were also made after addition of FCCP (1 μmoles/L), which depolarizes the mitochondrial membrane and induces maximal oxygen consumption. As shown, oxygen consumption rates were higher in PBMC’s from diabetic patients (P=0.01 for diabetes main effect and P=0.03 for diabetes by time interaction by repeated measures ANOVA).

Figure 2

Etomoxir reduces basal oxygen consumption rate (OCR) to an equivalent extent in diabetic patients and controls. Oxygen consumption rate was measured in PBMC’s incubated with the indicated concentrations of etomoxir as described in Methods. As shown, inhibiting fatty acid uptake produced a dose-dependent decrease in oxygen consumption (P<0.001) in both diabetic (n=5) and control subjects (n=5), but the response did not differ between groups (P=0.64 by analysis of variance for repeated measures).

Figure 3

Peak oxygen consumption in PBMC’s correlates with nitroglycerin-mediated dilation (upper panel), but not flow-mediated dilation (lower panel).