The mitochondrial ADP/ATP carrier (AAC, ANT), a member of the SLC25 family of solute carriers, plays a critical role in transporting purine nucleotides (ATP and ADP) as well as protons across the inner mitochondrial membrane. However, the precise mechanism and physiological significance of proton transport by ADP/ATP carrier remain unclear. Notably, the presence of uncouplers-such as long-chain fatty acids (FA) or artificial compounds like dinitrophenol (DNP)-is essential for this process. We explore two potential mechanisms that describe ADP/ATP carrier as either (i) a proton carrier that functions in the presence of FA or DNP, or (ii) an anion transporter (FA- or DNP). In the latter case, the proton is translocated by the neutral form of FA, which carries it from the matrix to the intermembrane space (FA-cycling hypothesis). Our recent results support this hypothesis. We describe a four-step mechanism for the "sliding" of the FA anion from the matrix to the mitochondrial intermembrane space and discuss a possible generalization of this mechanism to other SLC25 carriers.

• Keywords
• MD simulations, bilayer lipid membranes, membrane proteins, mitochondrial transporter, reconstituted protein, uncoupling protein,
• MeSH
• 2,4-Dinitrophenol metabolism   MeSH
• Adenosine Triphosphate metabolism   MeSH
• Biological Transport MeSH
• Ion Transport MeSH
• Humans MeSH
• Fatty Acids * metabolism   MeSH
• Mitochondrial ADP, ATP Translocases * metabolism   chemistry   MeSH
• Mitochondria * metabolism   MeSH
• Protons * MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Review MeSH
• Names of Substances
• 2,4-Dinitrophenol MeSH
• Adenosine Triphosphate MeSH
• Fatty Acids * MeSH
• Mitochondrial ADP, ATP Translocases * MeSH
• Protons * MeSH

Mitochondrial adenine nucleotide translocase (ANT) exchanges ADP for ATP to maintain energy production in the cell. Its protonophoric function in the presence of long-chain fatty acids (FA) is also recognized. Our previous results imply that proton/FA transport can be best described with the FA cycling model, in which protonated FA transports the proton to the mitochondrial matrix. The mechanism by which ANT1 transports FA anions back to the intermembrane space remains unclear. Using a combined approach involving measurements of the current through the planar lipid bilayers reconstituted with ANT1, site-directed mutagenesis and molecular dynamics simulations, we show that the FA anion is first attracted by positively charged arginines or lysines on the matrix side of ANT1 before moving along the positively charged protein-lipid interface and binding to R79, where it is protonated. We show that R79 is also critical for the competitive binding of ANT1 substrates (ADP and ATP) and inhibitors (carboxyatractyloside and bongkrekic acid). The binding sites are well conserved in mitochondrial SLC25 members, suggesting a general mechanism for transporting FA anions across the inner mitochondrial membrane.

• Keywords
• AAC, ADP/ATP carrier, arachidonic acid, fatty acid cycling hypothesis, fatty acids anion transport, proton transport, uncoupling proteins,
• MeSH
• Adenosine Triphosphate metabolism   MeSH
• Anions metabolism   MeSH
• Lipid Bilayers * MeSH
• Fatty Acids metabolism   MeSH
• Mitochondrial ADP, ATP Translocases metabolism   MeSH
• Protons * MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Adenosine Triphosphate MeSH
• Anions MeSH
• Lipid Bilayers * MeSH
• Fatty Acids MeSH
• Mitochondrial ADP, ATP Translocases MeSH
• Protons * MeSH

SIGNIFICANCE: Mitochondria are the energetic, metabolic, redox, and information signaling centers of the cell. Substrate pressure, mitochondrial network dynamics, and cristae morphology state are integrated by the protonmotive force Δp or its potential component, ΔΨ, which are attenuated by proton backflux into the matrix, termed uncoupling. The mitochondrial uncoupling proteins (UCP1-5) play an eminent role in the regulation of each of the mentioned aspects, being involved in numerous physiological events including redox signaling. Recent Advances: UCP2 structure, including purine nucleotide and fatty acid (FA) binding sites, strongly support the FA cycling mechanism: UCP2 expels FA anions, whereas uncoupling is achieved by the membrane backflux of protonated FA. Nascent FAs, cleaved by phospholipases, are preferential. The resulting Δp dissipation decreases superoxide formation dependent on Δp. UCP-mediated antioxidant protection and its impairment are expected to play a major role in cell physiology and pathology. Moreover, UCP2-mediated aspartate, oxaloacetate, and malate antiport with phosphate is expected to alter metabolism of cancer cells. CRITICAL ISSUES: A wide range of UCP antioxidant effects and participations in redox signaling have been reported; however, mechanisms of UCP activation are still debated. Switching off/on the UCP2 protonophoretic function might serve as redox signaling either by employing/releasing the extra capacity of cell antioxidant systems or by directly increasing/decreasing mitochondrial superoxide sources. Rapid UCP2 degradation, FA levels, elevation of purine nucleotides, decreased Mg2+, or increased pyruvate accumulation may initiate UCP-mediated redox signaling. FUTURE DIRECTIONS: Issues such as UCP2 participation in glucose sensing, neuronal (synaptic) function, and immune cell activation should be elucidated. Antioxid. Redox Signal. 29, 667-714.

• Keywords
• UCP2, anion transport, attenuation of superoxide formation, fatty acid cycling, mitochondrial uncoupling proteins, redox signaling,
• MeSH
• Antioxidants metabolism   MeSH
• Humans MeSH
• Mitochondrial Uncoupling Proteins metabolism   MeSH
• Oxidation-Reduction MeSH
• Signal Transduction * MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH
• Names of Substances
• Antioxidants MeSH
• Mitochondrial Uncoupling Proteins MeSH

AIMS: Pancreatic β-cell chronic lipotoxicity evolves from acute free fatty acid (FA)-mediated oxidative stress, unprotected by antioxidant mechanisms. Since mitochondrial uncoupling protein-2 (UCP2) plays antioxidant and insulin-regulating roles in pancreatic β-cells, we tested our hypothesis, that UCP2-mediated uncoupling attenuating mitochondrial superoxide production is initiated by FA release due to a direct H2O2-induced activation of mitochondrial phospholipase iPLA2γ. RESULTS: Pro-oxidant tert-butylhydroperoxide increased respiration, decreased membrane potential and mitochondrial matrix superoxide release rates of control but not UCP2- or iPLA2γ-silenced INS-1E cells. iPLA2γ/UCP2-mediated uncoupling was alternatively activated by an H2O2 burst, resulting from palmitic acid (PA) β-oxidation, and it was prevented by antioxidants or catalase overexpression. Exclusively, nascent FAs that cleaved off phospholipids by iPLA2γ were capable of activating UCP2, indicating that the previously reported direct redox UCP2 activation is actually indirect. Glucose-stimulated insulin release was not affected by UCP2 or iPLA2γ silencing, unless pro-oxidant activation had taken place. PA augmented insulin secretion via G-protein-coupled receptor 40 (GPR40), stimulated by iPLA2γ-cleaved FAs (absent after GPR40 silencing). INNOVATION AND CONCLUSION: The iPLA2γ/UCP2 synergy provides a feedback antioxidant mechanism preventing oxidative stress by physiological FA intake in pancreatic β-cells, regulating glucose-, FA-, and redox-stimulated insulin secretion. iPLA2γ is regulated by exogenous FA via β-oxidation causing H2O2 signaling, while FAs are cleaved off phospholipids, subsequently acting as amplifying messengers for GPR40. Hence, iPLA2γ acts in eminent physiological redox signaling, the impairment of which results in the lack of antilipotoxic defense and contributes to chronic lipotoxicity.

• MeSH
• Antioxidants pharmacology   MeSH
• Insulin-Secreting Cells drug effects   MeSH
• Group II Phospholipases A2 metabolism   MeSH
• Insulin metabolism   MeSH
• Ion Channels metabolism   MeSH
• Rats MeSH
• Lipids toxicity   MeSH
• Membrane Potential, Mitochondrial drug effects   MeSH
• Mitochondrial Proteins metabolism   MeSH
• Mitochondria drug effects   MeSH
• Cell Line, Tumor MeSH
• Oxidative Stress drug effects   MeSH
• Hydrogen Peroxide metabolism   MeSH
• Receptors, G-Protein-Coupled metabolism   MeSH
• Insulin Secretion MeSH
• Signal Transduction drug effects   MeSH
• Superoxides metabolism   MeSH
• tert-Butylhydroperoxide pharmacology   MeSH
• Uncoupling Protein 2 MeSH
• Animals MeSH
• Check Tag
• Rats MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Antioxidants MeSH
• Group II Phospholipases A2 MeSH
• G-protein-coupled receptor 40, rat MeSH Browser
• Insulin MeSH
• Ion Channels MeSH
• Lipids MeSH
• Mitochondrial Proteins MeSH
• Hydrogen Peroxide MeSH
• Receptors, G-Protein-Coupled MeSH
• Superoxides MeSH
• tert-Butylhydroperoxide MeSH
• Ucp2 protein, rat MeSH Browser
• Uncoupling Protein 2 MeSH

We reviewed mechanisms that determine reactive oxygen species (redox) homeostasis, redox information signaling and metabolic/regulatory function of autocrine insulin signaling in pancreatic β cells, and consequences of oxidative stress and dysregulation of redox/information signaling for their dysfunction. We emphasize the role of mitochondrion in β cell molecular physiology and pathology, including the antioxidant role of mitochondrial uncoupling protein UCP2. Since in pancreatic β cells pyruvate cannot be easily diverted towards lactate dehydrogenase for lactate formation, the respiration and oxidative phosphorylation intensity are governed by the availability of glucose, leading to a certain ATP/ADP ratio, whereas in other cell types, cell demand dictates respiration/metabolism rates. Moreover, we examine the possibility that type 2 diabetes mellitus might be considered as an inevitable result of progressive self-accelerating oxidative stress and concomitantly dysregulated information signaling in peripheral tissues as well as in pancreatic β cells. It is because the redox signaling is inherent to the insulin receptor signaling mechanism and its impairment leads to the oxidative and nitrosative stress. Also emerging concepts, admiting participation of redox signaling even in glucose sensing and insulin release in pancreatic β cells, fit in this view. For example, NADPH has been firmly established to be a modulator of glucose-stimulated insulin release.

• MeSH
• Insulin-Secreting Cells metabolism   pathology   MeSH
• Homeostasis * MeSH
• Insulin metabolism   MeSH
• Humans MeSH
• Mitochondria metabolism   MeSH
• Oxidation-Reduction MeSH
• Oxidative Stress MeSH
• Insulin Secretion MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH
• Names of Substances
• Insulin MeSH

Mitochondrial uncoupling proteins (UCPs) are pure anion uniporters, which mediate fatty acid (FA) uniport leading to FA cycling. Protonated FAs then flip-flop back across the lipid bilayer. An existence of pure proton channel in UCPs is excluded by the equivalent flux-voltage dependencies for uniport of FAs and halide anions, which are best described by the Eyring barrier variant with a single energy well in the middle of two peaks. Experiments with FAs unable to flip and alkylsulfonates also support this view. Phylogenetically, UCPs took advantage of the common FA-uncoupling function of SLC25 family carriers and dropped their solute transport function.

• MeSH
• Models, Biological MeSH
• Electrophoresis MeSH
• Ion Channels metabolism   MeSH
• Humans MeSH
• Mitochondrial Proteins metabolism   MeSH
• Protons MeSH
• Uncoupling Protein 1 MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH
• Research Support, N.I.H., Extramural MeSH
• Names of Substances
• Ion Channels MeSH
• Mitochondrial Proteins MeSH
• Protons MeSH
• Uncoupling Protein 1 MeSH

Existing controversies led us to analyze absolute mRNA levels of mitochondrial uncoupling proteins (UCP1-UCP5). Individual UCP isoform mRNA levels varied by up to four orders of magnitude in rat and mouse tissues. UCP2 mRNA content was relatively high (0.4 to 0.8 pg per 10 ng of total mRNA) in rat spleen, rat and mouse lung, and rat heart. Levels of the same order of magnitude were found for UCP3 mRNA in rat and mouse skeletal muscle, for UCP4 and UCP5 mRNA in mouse brain, and for UCP2 and UCP5 mRNA in mouse white adipose tissue. Significant differences in pattern were found for rat vs. mouse tissues, such as the dominance of UCP3/UCP5 vs. UCP2 transcript in mouse heart and vice versa in rat heart; or UCP2 (UCP5) dominance in rat brain contrary to 10-fold higher UCP4 and UCP5 dominance in mouse brain. We predict high antioxidant/antiapoptotic UCP function in tissues with higher UCP mRNA content.

• MeSH
• DNA Primers genetics   MeSH
• Species Specificity MeSH
• Ion Channels metabolism   MeSH
• Rats MeSH
• Membrane Transport Proteins metabolism   MeSH
• RNA, Messenger metabolism   MeSH
• Mitochondrial Uncoupling Proteins MeSH
• Mitochondrial Proteins metabolism   MeSH
• Brain metabolism   MeSH
• Myocardium metabolism   MeSH
• Mice MeSH
• Lung metabolism   MeSH
• Reverse Transcriptase Polymerase Chain Reaction MeSH
• Nerve Tissue Proteins metabolism   MeSH
• Spleen metabolism   MeSH
• Mitochondrial Membrane Transport Proteins MeSH
• Uncoupling Protein 2 MeSH
• Uncoupling Protein 3 MeSH
• Animals MeSH
• Check Tag
• Rats MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Comparative Study MeSH
• Names of Substances
• DNA Primers MeSH
• Ion Channels MeSH
• Membrane Transport Proteins MeSH
• RNA, Messenger MeSH
• Mitochondrial Uncoupling Proteins MeSH
• Mitochondrial Proteins MeSH
• Nerve Tissue Proteins MeSH
• Slc25a14 protein, rat MeSH Browser
• Slc25a27 protein, rat MeSH Browser
• Mitochondrial Membrane Transport Proteins MeSH
• Ucp2 protein, mouse MeSH Browser
• Ucp2 protein, rat MeSH Browser
• Ucp3 protein, mouse MeSH Browser
• Ucp3 protein, rat MeSH Browser
• Uncoupling Protein 2 MeSH
• Uncoupling Protein 3 MeSH