The diabetic phenotype is complex, requiring elucidation of key initiating defects. Diabetic myotubes express a primary reduced tricarboxylic acid (TCA) cycle flux but at present it is unclear in which part of the TCA cycle the defect is localised. In order to localise the defect we studied ATP production in isolated mitochondria from substrates entering the TCA cycle at various points. ATP production was measured by luminescence with or without concomitant ATP utilisation by hexokinase in mitochondria isolated from myotubes established from eight lean and eight type 2 diabetic subjects. The ATP production of investigated substrate combinations was significantly reduced in mitochondria isolated from type 2 diabetic subjects compared to lean. However, when ATP synthesis rates at different substrate combinations were normalized to the corresponding individual pyruvate-malate rate, there was no significant difference between groups. These results show that the primary reduced TCA cycle flux in diabetic myotubes is not explained by defects in specific part of the TCA cycle but rather results from a general downregulation of the TCA cycle.
Type 2 diabetes (T2D) is a disorder characterised by impaired insulin secretion from beta cells and insulin resistance (IR) in peripheral target tissues. Skeletal muscle is a key tissue site of the IR. Although a number of abnormalities have been identified in skeletal muscle from T2D subjects, the exact molecular mechanisms for IR have not been established. The diabetic phenotype is complex, requiring elucidation of key initiating defects. We have previously described that myotubes established from T2D subjects conserve the diabetic phenotype [
The intermediary metabolism of skeletal muscle
Eight lean and eight obese patients with type 2 diabetes participated in the study. Their clinical characteristics have been published [
Control | Diabetic | |
---|---|---|
8 | 8 | |
Age (years) | ||
Weight (kg) | ||
BMI (kg/m2) | ||
Fasting plasma glucose (mM) | ||
Fasting serum insulin (pM) | ||
Glucose infusion rate (mg/min) | ||
HbA1c (%) |
Data are means ± SE. *Significantly different from the controls
Cell cultures were established as previously described [
Mitochondria were isolated from cultured human myotubes using the MACS mitochondria isolation kit from Miltenyi Biotech, Germany [
The ATP synthesis in isolated mitochondria was measured by luminescence at baseline and during ATP utilization by the hexokinase reaction that catalyses the production of glucose 6-phosphate from glucose and ATP. The mitochondrial buffers contained 300 mM sucrose, 10 mM KCl, 10 mM KH2PO4, 10 mM Tris-Base, 0.1 mM EDTA, and 0.035 mM ADP supplemented with different substrate combinations. Initially we investigated the following combinations: 1 mM pyruvate and 1 mM malate (PM buffer), 1 mM malate and 5 mM glutamate (MG buffer), 5 mM succinate and 10
MitoTracker Green Probe (Molecular Probes, Eugene, OR) was used according to the manufacturer’s instructions. Fluorescence was determined on a VICTOR plate reader model 1420-050 (PerkinElmer, Finland) with excitation and emission wavelength of 485 and 535, respectively, as described previously, [
Data in text, tables, and figures are given as mean ± SEM. Statistical analyses were performed with SPSS (version 17.0). ANOVA test was used to assess significant differences between groups.
The ATP production in isolated mitochondria from diabetic myotubes was significantly reduced (
Rates of ATP synthesis in isolated mitochondria. The ATP synthesis rate was determined in isolated mitochondria of differentiated myotubes established from lean (open bars) and type 2 diabetic subjects (black bars). Isolated mitochondria were incubated with various substrates as indicated, supplemented with/without hexokinase as described in Section
The ATP synthesis in isolated mitochondria was measured at additional substrate combinations (acetate-malate (AM), citrate-malate (CM), isocitrate-malate (IM)). In order to show differences in substrate handling between mitochondria isolated from lean and obese T2D subjects ATP synthesis rates at different substrate combinations were normalized to values at PM. Mitochondria isolated from myotubes established from type 2 diabetic subjects did not reveal any differences in substrate handling for ATP synthesis compared to lean with or without the presence of hexokinase (
The mitochondrial mass in myotubes established from lean and obese diabetic subjects was measured in order to identify group differences in mitochondrial content. We could not detect any significant differences in mitochondrial mass between groups (
Mitochondrial mass. Mitochondrial mass was determined by MitoTracker Green fluorescence in myotubes established from lean (open bar) and T2D subjects (black bar). Data are means ± SE,
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 [
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 PKC
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 [
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 [
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 [
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.
Irene Lynfort, Jeanett Agergaard, and Ariane Minet provided excellent technical assistance. Kurt Højlund and Klaus Levin are thanked for muscle biopsies. The Odense University Hospital (free research funds) are thanked for financial support.