In this study, we demonstrated an efficient method to perform valid and reproducible measurements of mitochondrial mass and membrane potential in human mononuclear cells isolated from the peripheral venous circulation using JC-1 and NAO. Further, we demonstrated greater mitochondrial membrane polarization and lower mitochondrial mass in patients with T2DM compared to age matched controls. The presence of diabetes was the only subject characteristic associated with mitochondrial hyperpolarization and this association was independent of age. The relationship between mitochondrial mass and the presence of diabetes appears to be in part mediated by age and body mass index. We also found that mitochondrial superoxide production was higher in a subgroup of patients with T2DM relative to controls. Importantly, these data support our ability to make valid and reproducible measurements of mitochondrial homeostasis. To our knowledge, these data are the first to characterize the mitochondrial membrane potential and morphology in the mononuclear cells of patients with T2DM relative to non-diabetics. These findings add to the growing literature implicating derangements of mitochondrial homeostasis in the pathophysiology of type 2 diabetes.
We found mitochondrial hyperpolarization along with lower mitochondrial mass and spherical, less complex mitochondrial morphology in patients with T2DM relative to non-diabetics. Our findings parallel cell culture data delineating a similar pattern of mitochondrial hyperpolarization and morphological alterations central to the inflammatory activation process of monocytes and T-cells in type 2 diabetes.7, 23 12
Specifically, the hyperglycemic state of type 2 diabetes is known to trigger the production of multiple pro-inflammatory cytokines, induce cytotoxic and helper T-cell proliferation,24
and activate immune responses in T-cells and monocytes,25
and lead to concomitant increased ROS production.26, 27,28
The heightened overall inflammatory state in type 2 diabetes likely contributes significantly to the microvascular and macrovascular complications of this disease,29
suggesting our findings may have pathophysiological relevance to the cardiovascular morbidity of diabetes.
The present study also demonstrated significant differences in mitochondrial morphology in mononuclear cells from diabetic patients. There is a growing recognition that mitochondrial morphology and function are the significant linked.9, 10
With respect to diabetes, hyperglycemia induces rapid mitochondrial fission and this response is associated with excessive mitochondrial ROS production and mitochondrial hyperpolarization.12
Further, in vitro
cell culture work has demonstrated that increased mitochondrial fission and inhibition of mitochondrial fusion lead to less efficient ATP production,30
slower cell growth,31
and increased mitochondrial DNA damage.32
Our data extend these findings, suggesting chronic hyperglycemia and insulin resistance produce small, divided, and hyperpolarized mitochondria producing elevated levels of superoxide, contributing to an overall heightened state of activation and inflammation in mononuclear cells. The influence of diabetes on mitochondrial mass may be modified by both age and body mass, findings that merits future investigation.
In the present study, we used NAO fluorescence as an index of mitochondrial mass. This approach relies on a strong, direct relationship between mitochondrial cardiolipin and mitochondrial number. Cardiolipin is a large phospholipid specific to the mitochondrial inner membrane, comprising approximately 20% of membrane lipid content. Cardiolipin plays a key role in maintaining the fidelity of oxidative phosphorylation, but is prone to oxidative modifications and removal.33, 34
We cannot exclude reduced mitochondrial cardiolipin content in diabetic mononuclear cells as a contributor to the differences in NAO fluorescence observed. Given the importance of cardiolipin to mitochondrial membrane integrity, alterations in cardiolipin content in mononuclear cells in diabetics merit further investigation.
While useful for rapid assessments of structure and function, measurements of mitochondrial homeostasis using fluorescent probes should generally be considered semi-quantitative given limitations with all currently available probes.35
For example, studies using the mitochondrial membrane potential fluorophore TMRE may be problematic because it has been shown to bind to the inner mitochondrial membrane such that the fluorescent signal is primarily derived from bound rather than free TMRE depending on experimental conditions.36
With respect to probes employed in this study, NAO fluorescent intensity may be in part dependent on ΔΨm
These prior studies demonstrate that use of high-dose pharmacological agents to depolarize mitochondria also reduces NAO fluorescent intensity. While we cannot exclude ΔΨm
dependence of the NAO measurements in our study, these prior reports suggest that the difference in mitochondrial mass observed between non-diabetics and patients with diabetes in our study may actually be larger than we have observed using NAO to estimate mass.
JC-1 is widely used for qualitative estimates of ΔΨm
. Use of this probe to estimate membrane potential can be challenging given dose sensitivity of mitochondrial specificity of green fluorescence.35, 40
However, three lines of evidence suggest JC-1 measurements using our protocol represent a reasonable semi-quantitative measurement of mitochondrial membrane potential. First, we identified appropriate per-nuclear cellular localization JC-1 (). Second, our good reproducibility data suggest that, with our protocol, any non-specificity of monomeric JC-1 localization within a cell has limited effect on the variability of our measurements. This suggests our protocol supplies stable within-subjects cell loading concentrations of JC-1 to exposed mononuclear cells. Finally, our results are consistent with prior cell culture work showing mitochondrial hyperpolarization using JC-1 in the setting of hyperglycemia, adding to the validity of our measurements.41
While no probe for mitochondrial membrane potential measurement is without its limitations, including TMRE,42, 43
further verification of our membrane potential finding using a second mitochondrial-membrane potential sensitive fluorophore unrelated to JC-1 would help corroborate our findings.
Our study has several limitations. We did not measure glycosylated hemoglobin levels in our subjects. Future work looking at the relative effects of the acute glycemic milieu verses chronic glycemic control on our measurements of mitochondrial homeostasis will help discern the relative influences of these two exposures on these mitochondrial measurements. Due to the cross-sectional nature of this work, we cannot determine the relative causal contributions of impaired mitochondrial biogenesis, respiratory state ratio (state 3 vs. state 4) and hyperglycemia-induced fragmentation to our findings in patients with T2DM. Ethical considerations precluded our ability to study subjects withdrawn from their medications to determine their mitochondrial status in the absence of medications. Some of these medications, including metformin and HMG-CoA reductase inhibitors, may have relevant in vivo effects on mitochondria. The lowering LDL cholesterol concentrations in our T2DM population relative to non-diabetics is likely secondary to greater use of HMG-CoA reductase inhibitors by those with T2DM. The relative mitochondrial impact of medications on mitochondrial homeostasis will need to be further assessed. While JC-1's dependence on mitochondrial membrane potential is well-established, JC-1 aggregate formation can also be driven in part by mitochondrial volume. Given our finding of lower cell surface area of mitochondria in T2DM, it is conceivable a portion of our JC-1 findings may be secondary to lower mitochondrial volume in T2DM as suggested by our NAO studies and EM micrographs. Further studies manipulating the mitochondrial membrane potential while measuring mitochondrial superoxide production in both populations will be helpful to determine the relative contributions of differences in mitochondrial volume and membrane potential in our JC-1 observations. Balanced against these limitations are significant strengths including the novelty of establishing efficient and reproducible measurements of mitochondrial homeostasis in cells easily obtained by venipuncture and the application of this methodology to demonstrate potentially important differences in mitochondrial function and morphology between patients with T2DM and non-diabetics. These strengths underscore the potential of these measurements to be applied in future clinical research studies to better delineate the relevance of mitochondrial perturbations in type 2 diabetes and changes in mitochondrial function following interventions.