Mitochondria (mt) represent the vital hub of the molecular physiology of the cell, being decision-makers in cell life/death and information signaling, including major redox regulations and redox signaling. Now we review recent advances in understanding mitochondrial redox homeostasis, including superoxide sources and H2O2 consumers, i.e., antioxidant mechanisms, as well as exemplar situations of physiological redox signaling, including the intramitochondrial one and mt-to-cytosol redox signals, which may be classified as acute and long-term signals. This review exemplifies the acute redox signals in hypoxic cell adaptation and upon insulin secretion in pancreatic beta-cells. We also show how metabolic changes under these circumstances are linked to mitochondrial cristae narrowing at higher intensity of ATP synthesis. Also, we will discuss major redox buffers, namely the peroxiredoxin system, which may also promote redox signaling. We will point out that pathological thresholds exist, specific for each cell type, above which the superoxide sources exceed regular antioxidant capacity and the concomitant harmful processes of oxidative stress subsequently initiate etiology of numerous diseases. The redox signaling may be impaired when sunk in such excessive pro-oxidative state.

• MeSH
• Antioxidants metabolism   MeSH
• Insulin-Secreting Cells metabolism   MeSH
• Humans MeSH
• Mitochondria * metabolism   MeSH
• Oxidation-Reduction * MeSH
• Oxidative Stress physiology   MeSH
• Signal Transduction physiology   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Review MeSH
• Names of Substances
• Antioxidants MeSH

Aims: Glucose-stimulated insulin secretion (GSIS) in pancreatic β cells was expected to enhance mitochondrial superoxide formation. Hence, we elucidated relevant redox equilibria. Results: Unexpectedly, INS-1E cells at transitions from 3 (11 mM; pancreatic islets from 5 mM) to 25 mM glucose decreased matrix superoxide release rates (MitoSOX Red monitoring validated by MitoB) and H2O2 (mitoHyPer, subtracting mitoSypHer emission). Novel double-channel fluorescence lifetime imaging, approximating free mitochondrial matrix NADHF, indicated its ∼20% decrease. Matrix NAD+F increased on GSIS, indicated by the FAD-emission lifetime decrease, reflecting higher quenching of FAD by NAD+F. The participation of pyruvate/malate and pyruvate/citrate redox shuttles, elevating cytosolic NADPHF (iNAP1 fluorescence monitoring) at the expense of matrix NADHF, was indicated, using citrate (2-oxoglutarate) carrier inhibitors and cytosolic malic enzyme silencing: All changes vanished on these manipulations. 13C-incorporation from 13C-L-glutamine into 13C-citrate reflected the pyruvate/isocitrate shuttle. Matrix NADPHF (iNAP3 monitored) decreased. With decreasing glucose, the suppressor of Complex III site Q electron leak (S3QEL) suppressor caused a higher Complex I IF site contribution, but a lower superoxide fraction ascribed to the Complex III site IIIQo. Thus, the diminished matrix NADHF/NAD+F decreased Complex I flavin site IF superoxide formation on GSIS. Innovation: Mutually validated methods showed decreasing superoxide release into the mitochondrial matrix in pancreatic β cells on GSIS, due to the decreasing matrix NADHF/NAD+F (NADPHF/NADP+F) at increasing cytosolic NADPHF levels. The developed innovative methods enable real-time NADH/NAD+ and NADPH/NADP+ monitoring in any distinct cell compartment. Conclusion: The export of reducing equivalents from mitochondria adjusts lower mitochondrial superoxide production on GSIS, but it does not prevent oxidative stress in pancreatic β cells.

• Keywords
• Complex I, NADH/NAD+ ratio, fluorescence lifetime imaging, glucose-stimulated insulin secretion, mitochondrial superoxide generation, pancreatic β cells,
• MeSH
• Adenosine Triphosphate metabolism   MeSH
• Insulin-Secreting Cells metabolism   MeSH
• Cell Respiration MeSH
• Chromatography, Liquid MeSH
• Energy Metabolism MeSH
• Flavin-Adenine Dinucleotide metabolism   MeSH
• Glucose metabolism   MeSH
• Mass Spectrometry MeSH
• Rats MeSH
• Citric Acid metabolism   MeSH
• Membrane Potential, Mitochondrial MeSH
• Metabolic Networks and Pathways MeSH
• Metabolomics methods   MeSH
• Mitochondria metabolism   MeSH
• NAD metabolism   MeSH
• Hydrogen Peroxide metabolism   MeSH
• Insulin Secretion * MeSH
• Signal Transduction MeSH
• Superoxides metabolism   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
• Adenosine Triphosphate MeSH
• Flavin-Adenine Dinucleotide MeSH
• Glucose MeSH
• Citric Acid MeSH
• NAD MeSH
• Hydrogen Peroxide MeSH
• Superoxides MeSH

We have previously reported that transient knock-down of ATPase inhibitory factor 1 (IF1) by siRNA upregulates ATP levels and subsequently augments insulin secretion in model pancreatic β-cells INS-1E. Here we investigated how long-term IF1-overexpression impacts pancreatic β-cell bioenergetics and insulin secretion. We generated INS-1E cell line stably overexpressing native IF1. We revealed that IF1 overexpression leads to a substantial decrease in ATP levels and reduced glucose-stimulated insulin secretion. A decrease in total cellular ATP content was also reflected in decreased free ATP cytosolic and mitochondrial levels, as monitored with ATeam biosensor. Consistently, cellular respiration of IF1-overexpressing cells was decreased. 3D structured illumination microscopy (SIM) revealed a higher amount of insulin granules with higher volume in IF1-overexpressing cells. Similar effects occurred when cells were incubated at low glucose concentrations. Noteworthy, activation of PKA by dibutyryl cAMP entirely abolished the inhibitory effect of IF1 overexpression on ATP production and insulin secretion. Mitochondrial network morphology and cristae ultrastructure in INS-1E overexpressing IF1 remained mostly unchanged. Finally, we show that INS-1E cells decrease their IF1 protein levels relative to ATP synthase α-subunit in response to increased glucose. In conclusion, IF1 actively downregulates INS-1E cellular metabolism and reduces their ability to secrete insulin.

• MeSH
• Adenosine Triphosphate metabolism   MeSH
• Insulin-Secreting Cells metabolism   MeSH
• Cell Line MeSH
• Cyclic CMP analogs & derivatives   metabolism   MeSH
• Down-Regulation MeSH
• Glucose metabolism   MeSH
• Rats MeSH
• RNA, Small Interfering genetics   MeSH
• ATPase Inhibitory Protein MeSH
• Cyclic AMP-Dependent Protein Kinases metabolism   MeSH
• Proteins genetics   metabolism   MeSH
• Insulin Secretion * MeSH
• Signal Transduction MeSH
• Up-Regulation 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
• Adenosine Triphosphate MeSH
• Cyclic CMP MeSH
• dibutyryl cyclic-3',5'-cytidine monophosphate MeSH Browser
• Glucose MeSH
• RNA, Small Interfering MeSH
• Cyclic AMP-Dependent Protein Kinases MeSH
• Proteins 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

Reduced beta cell mass in pancreatic islets (PI) of Goto-Kakizaki (GK) rats is frequently observed in this diabetic model, but knowledge on delta cells is scarce. Aiming to compare delta cell physiology/pathology of GK to Wistar rats, we found that delta cell number increased over time as did somatostatin mRNA and delta cells distribution in PI is different in GK rats. Subtle changes in 6-week-old GK rats were found. With maturation and aging of GK rats, disturbed cytoarchitecture occurred with irregular beta cells accompanied by delta cell hyperplasia and loss of pancreatic polypeptide (PPY) positivity. Unlike the constant glucose-stimulation index for insulin PI release in Wistar rats, this index declined with GK age, whereas for somatostatin it increased with age. A decrease of GK rat PPY serum levels was found. GK rat body weight decreased with increasing hyperglycemia. Somatostatin analog octreotide completely blocked insulin secretion, impaired proliferation at low autocrine insulin, and decreased PPY secretion and mitochondrial DNA in INS-1E cells. In conclusion, in GK rats PI, significant local delta cell hyperplasia and suspected paracrine effect of somatostatin diminish beta cell viability and contribute to the deterioration of beta cell mass. Altered PPY-secreting cells distribution amends another component of GK PI's pathophysiology.

• MeSH
• Insulin Antagonists pharmacology   MeSH
• Insulin-Secreting Cells drug effects   metabolism   pathology   MeSH
• Somatostatin-Secreting Cells drug effects   metabolism   pathology   MeSH
• Diabetes Mellitus, Type 2 blood   metabolism   pathology   MeSH
• Hyperplasia MeSH
• Immunohistochemistry MeSH
• Rats, Inbred Strains MeSH
• Insulin metabolism   MeSH
• Insulin Resistance * MeSH
• RNA, Messenger metabolism   MeSH
• Cell Line, Tumor MeSH
• Octreotide pharmacology   MeSH
• Pancreatic Polypeptide antagonists & inhibitors   genetics   metabolism   MeSH
• Rats, Wistar MeSH
• Cell Proliferation drug effects   MeSH
• Insulin Secretion MeSH
• Somatostatin antagonists & inhibitors   genetics   metabolism   MeSH
• Aging * MeSH
• Cell Survival drug effects   MeSH
• Gene Expression Regulation, Developmental MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Insulin Antagonists MeSH
• Insulin MeSH
• RNA, Messenger MeSH
• Octreotide MeSH
• Pancreatic Polypeptide MeSH
• Somatostatin MeSH