Genistein regulates adipogenesis by blocking the function of adenine nucleotide translocase-2 in the mitochondria

Genistein exerts anti-adipogenic effects, but its target molecules remain unclear. Here, we delineated the molecular mechanism underlying the anti-adipogenic effect of genistein. A pulldown assay using genistein-immobilized beads identified adenine nucleotide translocase-2 as a genistein-binding protein in adipocytes. Adenine nucleotide translocase-2 exchanges ADP/ATP through the mitochondrial inner membrane. Similar to the knockdown of adenine nucleotide translocase-2, genistein treatment decreased ADP uptake into the mitochondria and ATP synthesis. Genistein treatment and adenine nucleotide translocase-2 knockdown suppressed adipogenesis and increased phosphorylation of AMP-activated protein kinase. Adenine nucleotide translocase-2 knockdown reduced the transcriptional activity of CCAAT/enhancer-binding protein β, whereas AMP-activated protein kinase inhibition restored the suppression of adipogenesis by adenine nucleotide translocase-2 knockdown. These results indicate that genistein interacts directly with adenine nucleotide translocase-2 to suppress its function. The downregulation of adenine nucleotide translocase-2 reduces the transcriptional activity of CCAAT/enhancer-binding protein β via activation of AMP-activated protein kinase, which consequently represses adipogenesis.

Mitochondria Isolated from Hearts Subjected to Ischemia/Reperfusion Benefit from Adenine Nucleotide Translocase 1 Overexpression

Reperfusion is the only feasible therapy following myocardial infarction, but reperfusion has been shown to damage mitochondrial function and disrupt energy production in the heart. Adenine nucleotide translocase 1 (ANT1) facilitates the transfer of ADP/ATP across the inner mitochondrial membrane; therefore, we tested whether ANT1 exerts protective effects on mitochondrial function during ischemia/reperfusion (I/R). The hearts of wild-type (WT) and transgenic ANT1-overexpressing (ANT1-TG) rats were exposed to I/R injury using the standard Langendorff technique, after which mitochondrial function, hemodynamic parameters, infarct size, and components of the contractile apparatus were determined. ANT1-TG hearts expressed higher ANT protein levels, with reduced levels of oxidative 4-hydroxynonenal ANT modifications following I/R. ANT1-TG mitochondria isolated from I/R hearts displayed stable calcium retention capacity (CRC) and improved membrane potential stability compared with WT mitochondria. Mitochondria isolated from ANT1-TG hearts experienced less restricted oxygen consumption than WT mitochondria after I/R. Left ventricular diastolic pressure (Pdia) decreased in ANT1-TG hearts compared with WT hearts following I/R. Preserved diastolic function was accompanied by a decrease in the phospho-lamban (PLB)/sarcoplasmic reticulum calcium ATPase (SERCA2a) ratio in ANT1-TG hearts compared with that in WT hearts. In addition, the phosphorylated (P)-PLB/PLB ratio increased in ANT1-TG hearts after I/R but not in WT hearts, which indicated more effective calcium uptake into the sarcoplasmic reticulum in ANT1-TG hearts. In conclusion, ANT1-TG rat hearts coped more efficiently with I/R than WT rat hearts, which was reflected by preserved mitochondrial energy balance, diastolic function, and calcium dynamics after reperfusion.

Adenine Nucleotide Translocase 1 Expression Modulates the Immune Response in Ischemic Hearts

Adenine nucleotide translocase 1 (ANT1) transfers ATP and ADP over the mitochondrial inner membrane and thus supplies the cell with energy. This study analyzed the role of ANT1 in the immune response of ischemic heart tissue. Ischemic ANT1 overexpressing hearts experienced a shift toward an anti-inflammatory immune response. The shift was characterized by low interleukin (IL)-1β expression and M1 macrophage infiltration, whereas M2 macrophage infiltration and levels of IL-10, IL-4, and transforming growth factor (TGFβ) were increased. The modulated immune response correlated with high mitochondrial integrity, reduced oxidative stress, low left ventricular end-diastolic heart pressure, and a high survival rate. Isolated ANT1-transgenic (ANT1-TG) cardiomyocytes expressed low levels of pro-inflammatory cytokines such as IL-1α, tumor necrosis factor α, and TGFβ. However, they showed increased expression and cellular release of anti-inflammatory immunomodulators such as vascular endothelial growth factor. The secretome from ANT1-TG cardiomyocytes initiated stress resistance when applied to ischemic wild-type cardiomyocytes and endothelial cells. It additionally prevented macrophages from expressing pro-inflammatory cytokines. Additionally, ANT1 expression correlated with genes that are related to cytokine and growth factor pathways in hearts of patients with ischemic cardiomyopathy. In conclusion, ANT1-TG cardiomyocytes secrete soluble factors that influence ischemic cardiac cells and initiate an anti-inflammatory immune response in ischemic hearts.

Mitochondrial Uncoupling Proteins (UCP1-UCP3) and Adenine Nucleotide Translocase (ANT1) Enhance the Protonophoric Action of 2,4-Dinitrophenol in Mitochondria and Planar Bilayer Membranes

2,4-Dinitrophenol (DNP) is a classic uncoupler of oxidative phosphorylation in mitochondria which is still used in “diet pills”, despite its high toxicity and lack of antidotes. DNP increases the proton current through pure lipid membranes, similar to other chemical uncouplers. However, the molecular mechanism of its action in the mitochondria is far from being understood. The sensitivity of DNP’s uncoupling action in mitochondria to carboxyatractyloside, a specific inhibitor of adenine nucleotide translocase (ANT), suggests the involvement of ANT and probably other mitochondrial proton-transporting proteins in the DNP’s protonophoric activity. To test this hypothesis, we investigated the contribution of recombinant ANT1 and the uncoupling proteins UCP1-UCP3 to DNP-mediated proton leakage using the well-defined model of planar bilayer lipid membranes. All four proteins significantly enhanced the protonophoric effect of DNP. Notably, only long-chain free fatty acids were previously shown to be co-factors of UCPs and ANT1. Using site-directed mutagenesis and molecular dynamics simulations, we showed that arginine 79 of ANT1 is crucial for the DNP-mediated increase of membrane conductance, implying that this amino acid participates in DNP binding to ANT1.

Adenine nucleotide translocase regulates airway epithelial metabolism, surface hydration and ciliary function

ABSTRACTAirway hydration and ciliary function are critical to airway homeostasis and dysregulated in chronic obstructive pulmonary disease (COPD), which is impacted by cigarette smoking and has no therapeutic options. We utilized a high-copy cDNA library genetic selection approach in the amoeba Dictyostelium discoideum to identify genetic protectors to cigarette smoke. Members of the mitochondrial ADP/ATP transporter family adenine nucleotide translocase (ANT) are protective against cigarette smoke in Dictyostelium and human bronchial epithelial cells. Gene expression of ANT2 is reduced in lung tissue from COPD patients and in a mouse smoking model, and overexpression of ANT1 and ANT2 resulted in enhanced oxidative respiration and ATP flux. In addition to the presence of ANT proteins in the mitochondria, they reside at the plasma membrane in airway epithelial cells and regulate airway homeostasis. ANT2 overexpression stimulates airway surface hydration by ATP and maintains ciliary beating after exposure to cigarette smoke, both of which are key functions of the airway. Our study highlights a potential for upregulation of ANT proteins and/or of their agonists in the protection from dysfunctional mitochondrial metabolism, airway hydration and ciliary motility in COPD.This article has an associated First Person interview with the first author of the paper.

Interaction of Agaric Acid with the Adenine Nucleotide Translocase Induces Mitochondrial Oxidative Stress

Mitochondrial permeability transition is characterized by the opening of a transmembranal pore that switches membrane permeability from specific to nonspecific. This structure allows the free traffic of ions, metabolites, and water across the mitochondrial inner membrane. The opening of the permeability transition pore is triggered by oxidative stress along with calcium overload. In this work, we explored if oxidative stress is a consequence, rather than an effector of the pore opening, by evaluating the interaction of agaric acid with the adenine nucleotide translocase, a structural component of the permeability transition pore. We found that agaric acid induces transition pore opening, increases the generation of oxygen-derived reactive species, augments the oxidation of unsaturated fatty acids in the membrane, and promotes the detachment of cytochrome c from the inner membrane. The effect of agaric acid was inhibited by the antioxidant tamoxifen in association with decreased binding of the thiol reagent eosin-3 maleimide to the adenine nucleotide translocase. We conclude that agaric acid promotes the opening of the pore, increasing ROS production that exerts oxidative modification of critical thiols in the adenine nucleotide translocase.