Platelet mitochondrial dysfunction is evident in type 2 diabetes in association with modifications of mitochondrial anti-oxidant stress proteins

Abstract

Objective: Mitochondrial dysfunction and oxidative stress in insulin responsive tissues is implicated in the pathogenesis of type 2 diabetes. Whether these perturbations extend to other tissues and contribute to their pathophysiology is less well established. The objective of this study was to investigate platelet mitochondria to evaluate whether type 2 diabetes associated mitochondrial dysfunction is evident in circulating cells.

Method: A pilot study of mitochondrial respiratory function and proteomic changes comparing platelets extracted from insulin sensitive (n=8) and type 2 diabetic subjects (n=7).

Results: In-situ platelet mitochondria show diminished oxygen consumption and lower oxygen-dependent ATP synthesis in diabetic vs. control subjects. Mass spectrometric identification and confirmatory immunoblot analysis identifies induction of the mitochondrial anti-oxidant enzymes superoxide dismutase 2 and thioredoxin-dependent peroxide reductase 3 in platelets of diabetic subjects. As oxidative stress upregulates anti-oxidant enzymes we assessed mitochondrial protein carbonylation as an index of oxidative-stress. Platelets of diabetic subjects exhibit significantly increased protein carbonylation compared to controls.

Conclusions: As platelets are anuclear fragments of megakaryocytes, our data suggest that the bone marrow compartment in type 2 diabetic subjects is exposed to increased mitochondrial oxidative stress with upregulation of nuclear-encoded antioxidant mitochondrial enzymes. This 'stress-signature' in platelets of diabetic subjects is associated with a diminution of their mitochondrial contribution to energy production and support that mitochondrial perturbations in type 2 diabetes extends beyond the classical insulin responsive tissues. Platelets, as "accessible human tissue", may be useful to measure the mitochondrial modulatory effects of emerging anti-diabetic therapeutics.

Fig. 1.

Mitochondrial respiration is perturbed in diabetic subject platelets. A. Representative tracing of basal oxygen consumption and maximal oxygen consumption induced by the uncoupler dinitrophenol comparing platelets from a control and diabetic subject. B. Representative tracing of oxygen consumption in response to inhibition of the F₁/F_o ATP synthase with oligomycin, and in response to the subsequent inhibition of electron transfer chain activity with the inhibitors rotenone and antimycin A. The reduction in oxygen consumption following oligomycin represents oxygen utilization for ATP production. The difference in oxygen consumption between the two pharmacologic interventions represents the proton leak, and the residual oxygen utilization represents cytosolic oxygen consumption. C. Histograms showing the absolute differences in oxygen consumption comparing groups normalized to total protein content. The asterisk represents a p<0.05 versus respective control subjects. Abbreviation: Mt - Mitochondria

Fig. 2.

Protein analyses showing altered reactive oxygen species regulatory proteins and consequences in diabetic subject platelets. A. Magnification of 2D-DIGE with Progenesis Samespot software after mass spectrometry which located and analyzed protein spots assigning statistical confidence and difference in anti-oxidant protein levels. B. Post-translation modification of SOD2 on 2D-DIGE that would be compatible with change in the phosphorylation status between control and diabetic samples. C. Immunoblot confirmation of expression of anti-oxidant proteins. β-actin shows protein loading control. D. Histogram shows protein expression levels from all subjects. E. Relative carbonylation levels from platelet mitochondrial proteins from each subject. Asterisks highlight p<0.05 versus relative control subjects.