Pyruvate, acetate, malate, citrate, isocitrate, glutamate, and succinate enter the TCA cycle at different sites before they are oxidised, creating a transmembrane potential, powering the phosphorylation of ADP to ATP. Their energy is predominantly transferred to complex I of the electron transport chain (ECT) for all substrates except for the combination succinate-rotenone which preliminary deliver to complex II. As in our previous studies of ATP production in isolated mitochondria from diabetic myotubes, the ATP production was reduced compared to mitochondria from lean [13
]. To search for TCA cycle defects in diabetic mitochondria, we exposed isolated mitochondria for substrates with different TCA cycle entries to locate TCA cycle defects. All entrance points to the TCA cycle gave similar normalised ATP synthesis rates in the two groups indicating that there may not be a major single primary defect present in the TCA cycle and the reduced TCA cycle flux in diabetic mitochondria seems to be based on a general TCA cycle downregulation. Both complex I and II substrates gave the same results indicating that the defect is either downstream from complex II, that is, at the sites of cytochrome C oxidase or ATP synthase, or a general reduction in TCA cycle flux. Recent evidence from global approaches such as microarray gene expression analysis has suggested that the basis for reduced mitochondrial ATP production in skeletal muscle from T2D could be a functional impairment in oxidative phosphorylation (OXPHOS) [23
]. However, transcriptional profiling of myotubes established from T2D subjects did not show evidence of a primary defect in OXPHOS genes [22
]. Moreover, we have previously determined the uncoupled respiration rate in myotubes established from lean, obese, and T2D subjects and found no significant differences between groups, suggesting that the ETC did not express intrinsic defects [12
]. Contradicting a primary major defect in the oxidative phosphorylation in human myotubes, the basal glucose oxidation decreased when oxidative phosphorylation was either inhibited by Antimycin A (Complex III inhibitor) or oligomycin (F0-F1 inhibitor) in myotubes established from lean, obese, and T2D subjects [6
] showing that lean, obese, and diabetic react in the same manner. However, diabetic myotubes express an increased basal glucose oxidation during normophysiological condition and still expressed an increased basal glucose oxidation compared to lean myotubes, despite inhibition of the oxidative phosphorylation, indicating that defects in the oxidative phosphorylation were not the major mechanism to explain primary changes in diabetic myotube metabolism [6
]. To identify and quantify changes in protein abundance between human myotubes obtained from lean, obese, and type 2 diabetic subjects we did a quantitative proteomic study [15
]. Despite a clear diabetic phenotype in diabetic myotubes, only twelve proteins were differentially expressed between the three different groups. Proteins from all major pathways known to be important in T2D were well characterized including the TCA cycle, lipid oxidation, oxidative phosphorylation, the glycolytic pathway, and glycogen metabolism. None of these enzymes were found to be regulated at the level of protein expression or degradation, suggesting that above differences in ATP synthesis may not be found on the protein expression level. Taken together, the above data indicates that there are no intrinsic single defects in the TCA cycle which could explain the reduced TCA cycle flux in diabetic mitochondria.
A general downregulation of the TCA cycle can be obtained at the level of posttranslational modification (PTM) of TCA cycle proteins or through changes in intramyocellular energy-redox state controlling the overall TCA cycle flux rate. Less is known about mitochondrial phosphatases and kinases. Studies indicate that cytosolic kinases may be translocated into the mitochondria that is, PKA, PKC, or AKT. A recent paper reports that the activity of mitochondrial aconitase (a TCA cycle enzyme) in type 1 diabetic rat hearts is regulated by PKCβ2
, whose activity is dependent on the degree of phosphorylation [25
]. Augmented phosphorylation of mitochondrial aconitase was associated with a reduced TCA cycle flux, based on an increased reverse activity, while the forward reaction was normal. That PTM could be part of the explanation for a reduced TCA cycle flux in diabetic mitochondria is supported by our previous finding of a 14% reduced citrate synthase activity in diabetic myotubes [12
] which cannot be explained by changes in protein expression [15
]. Additional phosphoproteomic studies are required to test whether the impaired TCA cycle flux, in diabetic mitochondria, is based on posttranscriptional modifications of TCA cycle enzymes.
A more overall regulation of the TCA cycle in human myotubes may be based on the mitochondrial matrix phosphorylation potential (Pi + ADP/ATP) and the pyridine nucleotide redox poise and concentration (NADH/NAD+). Increasing substrate availability is followed by increasing concentrations of reduction equivalents and ATP which, through allosteric inhibition, can downregulate the TCA cycle flux. Previously we determined the energy charge, the level of ATP, ADP, and AMP in myotubes established from lean, obese, and obese type 2 diabetic subjects at normophysiological conditions and could not verify differences between groups [26
] indicating that the energy state may not account for differences in the TCA cycle flux between groups.
Increased oxidative stress has been implicated in the development of insulin resistance in type 2 diabetes by both indirect and direct evidence based on increased damage of DNA, lipids, and proteins [27
]. Recently we measured hydrogen peroxide production/mitochondrial mass and found it significantly reduced in diabetic myotubes compared to lean controls indicating that an increased ROS production may not be the main mechanism accounting for a reduced TCA flux in diabetic mitochondria [16
The reduced but similar ATP production on different substrates in diabetic mitochondria could point to reduced substrate availability as explanation for obtained differences, that is, based on impaired transport/interchange of substrates across the inner mitochondrial membrane. Several substrates were used in the present study requiring different transporters indicating that transport/interchange of substrate and intermediates could be generally impaired in diabetic mitochondria. However, we have recently described that the inner mitochondrial membrane potential was conserved in diabetic mitochondria [16
]. Further studies are required to clarify whether substrate availability may be part of the responsible mechanism for a reduced ATP production in diabetic mitochondria.
An overall reduced TCA flux in diabetic myotubes compared to lean myotubes could be based on a reduced mitochondrial mass in diabetic myotubes. We addressed the question by measuring the mitochondrial mass in myotubes established from lean and type 2 diabetic subjects and could not show significant differences between groups.
In summary, we tested the hypothesis that the reduced TCA cycle flux in diabetic mitochondria was based on site-specific TCA cycle defects but we could not find evidence for this. The primary reduced TCA cycle flux in diabetic myotubes is explained by a general downregulation of the TCA cycle. We hypothesize that the impaired TCA cycle flux, in diabetic mitochondria, is based on posttranscriptional modifications of TCA cycle enzymes.