Modular Kinetic Analysis of the Adenine Nucleotide Translocator–Mediated Effects of Palmitoyl-CoA on the Oxidative Phosphorylation in Isolated Rat Liver Mitochondria Jolita Ciapaite, 1 Gerco Van Eikenhorst, 1 Stephan J.L. Bakker, 2 Michaela Diamant, 3 Robert J. Heine, 3 Marijke J. Wagner, 1 Hans V. Westerhoff, 1 and Klaas Krab 1 To test whether long-chain fatty acyl-CoA esters link obesity with type 2 diabetes through inhibition of the mitochondrial adenine nucleotide translocator, we ap- plied a system-biology approach, dual modular kinetic analysis, with mitochondrial membrane potential () and the fraction of matrix ATP as intermediates. We found that 5 mol/l palmitoyl-CoA inhibited adenine nucleotide translocator, without direct effect on other components of oxidative phosphorylation. Indirect ef- fects depended on how oxidative phosphorylation was regulated. When the electron donor and phosphate ac- ceptor were in excess, and the mitochondrial “work” flux was allowed to vary, palmitoyl-CoA decreased phos- phorylation flux by 38% and the fraction of ATP in the medium by 39%.  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:  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,  and the fraction of ATP were not af- fected. Palmitoyl-CoA increased the extramitochondrial AMP concentration significantly. The effects of palmi- toyl-CoA in our model system support the proposed mechanism linking obesity and type 2 diabetes through an effect on adenine nucleotide translocator. Diabetes 54:944 –951, 2005 O besity is a common finding in patients with type 2 diabetes (1–3). Numerous studies suggest that the oversupply of lipid to nonadipose tissues might result in lipotoxicity and contrib- ute to the development of the insulin resistance syndrome and type 2 diabetes (4,5). The molecular mechanisms responsible for lipotoxicity, insulin resistance, and -cell dysfunction are not fully understood. It is particularly confusing that so many processes appear to be affected at the same time, but not always to the same extent, and often in paradoxical ways. It has been hypothesized that inhibition of the mitochon- drial adenine nucleotide translocator by long-chain fatty acyl-CoA esters, the active form of fatty acids, may be an important link between obesity and type 2 diabetes (6,7). Adenine nucleotide translocator is an enzyme that cata- lyzes the exchange of mitochondrial ATP for cytosolic ADP (8). It was shown that long-chain fatty acyl-CoA esters are potent inhibitors of adenine nucleotide translo- cator from both the cytosolic and the matrix side of the inner mitochondrial membrane (9). It was proposed that accumulation of long-chain fatty acyl-CoA esters in the cell and subsequent inhibition of adenine nucleotide translo- cator could lead to an increase in the matrix ATP-to-ADP ratio, membrane potential (; i.e., electric potential across the inner mitochondrial membrane [out minus in]), and oxygen free radical production. In -cells, like in other cells (10), an initial increase in O 2   (superoxide anion radical) production could stimulate cell growth and con- tribute to compensatory hyperinsulinemia. Later on, sus- tained high levels of O 2   should result in a decline in -cell function and viability. Moreover, the inhibition of adenine nucleotide translocator was proposed to decrease the cytosolic ATP-to-ADP ratio and shift the equilibrium of the adenylate kinase toward production of ATP and AMP. The increase in AMP concentration should promote for- mation of adenosine, which in turn was reported to have both inhibitory and stimulatory effects on insulin-depen- dent glucose uptake (11–13). It was hypothesized that an increased extracellular adenosine concentration could ex- plain such pathophysiological features of type 2 diabetes as elevated levels of uric acid, increased sympathetic activity, and expansion of extracellular volume (9). For From the 1 Department of Molecular Cell Physiology, Institute for Molecular Cell Biology, BioCenter Amsterdam, Faculty of Earth and Life Sciences, Vrije Universiteit, Amsterdam, the Netherlands; the 2 Department of Internal Medi- cine, University Hospital Groningen, Groningen, the Netherlands; and the 3 Department of Endocrinology, Institute for Cardiovascular Research, Vrije Universiteit Medical Center, Amsterdam, the Netherlands. Address correspondence and reprint requests to Klaas Krab, Department of Molecular Cell Physiology, Institute for Molecular Cell Biology, BioCenter Amsterdam, Faculty of Earth and Life Sciences, Vrije Universiteit, De Boele- laan 1085, NL-1081 HV Amsterdam, the Netherlands. E-mail: klaas@bio.vu.nl. Received for publication 6 July 2004 and accepted in revised form 4 January 2005. Ap5A, P 1 P 5 -di(adenosine-5) pentaphosphate; TPP  , tetraphenylphospho- nium ion. © 2005 by the American Diabetes Association. 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