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We proposed that inhibition of mitochondrial adenine nucleotide translocator (ANT) by long chain acyl-CoA (LCAC) underlies the mechanism associating obesity and type 2 diabetes. Here we test that after long-term exposure to a high-fat diet (HFD): (i) there is no adaptation of the mitochondrial compartment that would hinder such ANT inhibition, and (ii) ANT has significant control of the relevant aspects of oxidative phosphorylation. After 7 weeks, HFD induced a 24+/-6% increase in hepatic LCAC concentration and accumulation of the oxidative stress marker N(epsilon)-(carboxymethyl)lysine. HFD did not significantly affect mitochondrial copy number, oxygen uptake, membrane potential (Deltapsi), ADP/O ratio, and the content of coenzyme Q(9), cytochromes b and a+a(3). Modular kinetic analysis showed that the kinetics of substrate oxidation, phosphorylation, proton leak, ATP-production and ATP-consumption were not influenced significantly. After HFD-feeding ANT exerted considerable control over oxygen uptake (control coefficient C=0.14) and phosphorylation fluxes (C=0.15), extra- (C=0.23) and intramitochondrial (C=-0.56) ATP/ADP ratios, and Deltapsi (C=-0.11). We conclude that although HFD induces accumulation of LCAC and N(epsilon)-(carboxymethyl)lysine, oxidative phosphorylation does not adapt to these metabolic challenges. Furthermore, ANT retains control of fluxes and intermediates, making inhibition of this enzyme a more probable link between obesity and type 2 diabetes.

Inhibition of the mitochondrial adenine nucleotide translocator (ANT) by long-chain acyl-CoA esters has been proposed to contribute to cellular dysfunction in obesity and type 2 diabetes by increasing formation of reactive oxygen species and adenosine via effects on the coenzyme Q redox state, mitochondrial membrane potential (Deltapsi) and cytosolic ATP concentrations. We here show that 5 microm palmitoyl-CoA increases the ratio of reduced to oxidized coenzyme Q (QH(2)/Q) by 42 +/- 9%, Deltapsi by 13 +/- 1 mV (9%), and the intramitochondrial ATP/ADP ratio by 352 +/- 34%, and decreases the extramitochondrial ATP/ADP ratio by 63 +/- 4% in actively phosphorylating mitochondria. The latter reduction is expected to translate into a 24% higher extramitochondrial AMP concentration. Furthermore, palmitoyl-CoA induced concentration-dependent H(2)O(2) formation, which can only partly be explained by its effect on Deltapsi. Although all measured fluxes and intermediate concentrations were affected by palmitoyl-CoA, modular kinetic analysis revealed that this resulted mainly from inhibition of the ANT. Through Metabolic Control Analysis, we then determined to what extent the ANT controls the investigated mitochondrial properties. Under steady-state conditions, the ANT moderately controlled oxygen uptake (control coefficient C = 0.13) and phosphorylation (C = 0.14) flux. It controlled intramitochondrial (C = -0.70) and extramitochondrial ATP/ADP ratios (C = 0.23) more strongly, whereas the control exerted over the QH(2)/Q ratio (C = -0.04) and Deltapsi (C = -0.01) was small. Quantitative assessment of the effects of palmitoyl-CoA showed that the mitochondrial properties that were most strongly controlled by the ANT were affected the most. Our observations suggest that long-chain acyl-CoA esters may contribute to cellular dysfunction in obesity and type 2 diabetes through effects on cellular energy metabolism and production of reactive oxygen species.

To test whether long-chain fatty acyl-CoA esters link obesity with type 2 diabetes through inhibition of the mitochondrial adenine nucleotide translocator, we applied a system-biology approach, dual modular kinetic analysis, with mitochondrial membrane potential (Deltapsi) and the fraction of matrix ATP as intermediates. We found that 5 mumol/l palmitoyl-CoA inhibited adenine nucleotide translocator, without direct effect on other components of oxidative phosphorylation. Indirect effects depended on how oxidative phosphorylation was regulated. When the electron donor and phosphate acceptor were in excess, and the mitochondrial "work" flux was allowed to vary, palmitoyl-CoA decreased phosphorylation flux by 38% and the fraction of ATP in the medium by 39%. Deltapsi increased by 15 mV, and the fraction of matrix ATP increased by 46%. Palmitoyl-CoA had a stronger effect when the flux through the mitochondrial electron transfer chain was maintained constant: Deltapsi increased by 27 mV, and the fraction of matrix ATP increased 2.6 times. When oxidative phosphorylation flux was kept constant by adjusting the rate using hexokinase, Deltapsi and the fraction of ATP were not affected. Palmitoyl-CoA increased the extramitochondrial AMP concentration significantly. The effects of palmitoyl-CoA in our model system support the proposed mechanism linking obesity and type 2 diabetes through an effect on adenine nucleotide translocator.