ATPase Inhibitory factor 1 (IF1) is an endogenous regulator of mitochondrial ATP synthase, which is involved in cellular metabolism. Although great progress has been made, biological roles of IF1 and molecular mechanisms of its action are still to be elucidated. Here, we show that IF1 is present in pancreatic β-cells, bound to the ATP synthase also under normal physiological conditions. IF1 silencing in model pancreatic β-cells (INS-1E) increases insulin secretion over a range of glucose concentrations. The left-shifted dose-response curve reveals excessive insulin secretion even under low glucose, corresponding to fasting conditions. A parallel increase in cellular respiration and ATP levels is observed. To conclude, our results indicate that IF1 is a negative regulator of insulin secretion involved in pancreatic β-cell glucose sensing.

• MeSH
• beta-buňky cytologie   metabolismus   MeSH
• glukosa metabolismus   MeSH
• inzulin metabolismus   MeSH
• krysa rodu rattus MeSH
• nádorové buněčné linie MeSH
• potkani Wistar MeSH
• proteiny metabolismus   MeSH
• spotřeba kyslíku fyziologie   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• zvířata MeSH
• Publikační typ
• dopisy MeSH
• práce podpořená grantem 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
• adenosintrifosfát metabolismus   MeSH
• beta-buňky metabolismus   MeSH
• buněčné linie MeSH
• CMP cyklický analogy a deriváty   metabolismus   MeSH
• down regulace MeSH
• glukosa metabolismus   MeSH
• krysa rodu rattus MeSH
• malá interferující RNA genetika   MeSH
• proteinkinasy závislé na cyklickém AMP metabolismus   MeSH
• proteiny genetika   metabolismus   MeSH
• sekrece inzulinu * MeSH
• signální transdukce MeSH
• up regulace MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Hydrolysis of ATP by the mitochondrial F-ATPase is inhibited by a protein called IF1 . In the parasitic flagellate, Trypanosoma brucei, this protein, known as TbIF1 , is expressed exclusively in the procyclic stage, where the F-ATPase is synthesizing ATP. In the bloodstream stage, where TbIF1 is absent, the F-ATPase hydrolyzes ATP made by glycolysis and compensates for the absence of a proton pumping respiratory chain by translocating protons into the intermembrane space, thereby maintaining the essential mitochondrial membrane potential. We have defined regions and amino acid residues of TbIF1 that are required for its inhibitory activity by analyzing the binding of several modified recombinant inhibitors to F1 -ATPase isolated from the procyclic stage of T. brucei. Kinetic measurements revealed that the C-terminal portion of TbIF1 facilitates homodimerization, but it is not required for the inhibitory activity, similar to the bovine and yeast orthologs. However, in contrast to bovine IF1 , the inhibitory capacity of the C-terminally truncated TbIF1 diminishes with decreasing pH, similar to full length TbIF1 . This effect does not involve the dimerization of active dimers to form inactive tetramers. Over a wide pH range, the full length and C-terminally truncated TbIF1 form dimers and monomers, respectively. TbIF1 has no effect on bovine F1 -ATPase, and this difference in the mechanism of regulation of the F-ATPase between the host and the parasite could be exploited in the design of drugs to combat human and animal African trypanosomiases.

• MeSH
• inhibitory enzymů chemie   farmakologie   MeSH
• mutace MeSH
• proteiny chemie   genetika   farmakologie   MeSH
• protonové ATPasy antagonisté a inhibitory   MeSH
• regulace genové exprese * MeSH
• sekvence aminokyselin MeSH
• sekvenční homologie MeSH
• skot MeSH
• Trypanosoma brucei brucei enzymologie   MeSH
• zvířata MeSH
• Check Tag
• skot MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The mitochondrial (mt) FoF1-ATP synthase of the digenetic parasite, Trypanosoma brucei, generates ATP during the insect procyclic form (PF), but becomes a perpetual consumer of ATP in the mammalian bloodstream form (BF), which lacks a canonical respiratory chain. This unconventional dependence on FoF1-ATPase is required to maintain the essential mt membrane potential (Δψm). Normally, ATP hydrolysis by this rotary molecular motor is restricted to when eukaryotic cells experience sporadic hypoxic conditions, during which this compulsory function quickly depletes the cellular ATP pool. To protect against this cellular treason, the highly conserved inhibitory factor 1 (IF1) binds the enzyme in a manner that solely inhibits the hydrolytic activity. Intriguingly, we were able to identify the IF1 homolog in T. brucei (TbIF1), but determined that its expression in the mitochondrion is tightly regulated throughout the life cycle as it is only detected in PF cells. TbIF1 appears to primarily function as an emergency brake in PF cells, where it prevented the restoration of the Δψm by FoF1-ATPase when respiration was chemically inhibited. In vitro, TbIF1 overexpression specifically inhibits the hydrolytic activity but not the synthetic capability of the FoF1-ATP synthase in PF mitochondria. Furthermore, low μM amounts of recombinant TbIF1 achieve the same inhibition of total mt ATPase activity as the FoF1-ATPase specific inhibitors, azide and oligomycin. Therefore, even minimal ectopic expression of TbIF1 in BF cells proved lethal as the indispensable Δψm collapsed due to inhibited FoF1-ATPase. In summary, we provide evidence that T. brucei harbors a natural and potent unidirectional inhibitor of the vital FoF1-ATPase activity that can be exploited for future structure-based drug design.

• MeSH
• adenosintrifosfát metabolismus   MeSH
• hydrolýza MeSH
• inhibitory enzymů metabolismus   MeSH
• protonové ATPasy metabolismus   MeSH
• regulace genové exprese * MeSH
• Trypanosoma brucei brucei enzymologie   genetika   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH

Significance: Mitochondria determine glucose-stimulated insulin secretion (GSIS) in pancreatic β-cells by elevating ATP synthesis. As the metabolic and redox hub, mitochondria provide numerous links to the plasma membrane channels, insulin granule vesicles (IGVs), cell redox, NADH, NADPH, and Ca2+ homeostasis, all affecting insulin secretion. Recent Advances: Mitochondrial redox signaling was implicated in several modes of insulin secretion (branched-chain ketoacid [BCKA]-, fatty acid [FA]-stimulated). Mitochondrial Ca2+ influx was found to enhance GSIS, reflecting cytosolic Ca2+ oscillations induced by action potential spikes (intermittent opening of voltage-dependent Ca2+ and K+ channels) or the superimposed Ca2+ release from the endoplasmic reticulum (ER). The ATPase inhibitory factor 1 (IF1) was reported to tune the glucose sensitivity range for GSIS. Mitochondrial protein kinase A was implicated in preventing the IF1-mediated inhibition of the ATP synthase. Critical Issues: It is unknown how the redox signal spreads up to the plasma membrane and what its targets are, what the differences in metabolic, redox, NADH/NADPH, and Ca2+ signaling, and homeostasis are between the first and second GSIS phase, and whether mitochondria can replace ER in the amplification of IGV exocytosis. Future Directions: Metabolomics studies performed to distinguish between the mitochondrial matrix and cytosolic metabolites will elucidate further details. Identifying the targets of cell signaling into mitochondria and of mitochondrial retrograde metabolic and redox signals to the cell will uncover further molecular mechanisms for insulin secretion stimulated by glucose, BCKAs, and FAs, and the amplification of secretion by glucagon-like peptide (GLP-1) and metabotropic receptors. They will identify the distinction between the hub β-cells and their followers in intact and diabetic states. Antioxid. Redox Signal. 36, 920-952.

• MeSH
• adenosintrifosfát metabolismus   MeSH
• beta-buňky * metabolismus   MeSH
• glukosa metabolismus   MeSH
• inzulin metabolismus   MeSH
• Langerhansovy ostrůvky * metabolismus   MeSH
• mitochondrie metabolismus   MeSH
• NAD metabolismus   MeSH
• NADP metabolismus   MeSH
• sekrece inzulinu MeSH
• sekretagoga metabolismus   MeSH
• vápník metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH

The central stalk of mitochondrial ATP synthase consists of subunits γ, δ, and ε, and along with the membraneous subunit c oligomer constitutes the rotor domain of the enzyme. Our previous studies showed that mutation or deficiency of ε subunit markedly decreased the content of ATP synthase, which was otherwise functionaly and structuraly normal. Interestingly, it led to accumulation of subunit c aggregates, suggesting the role of the ε subunit in assembly of individual enzyme domains. In the present study we focused on the role of subunits γ and δ. Using shRNA knockdown in human HEK293 cells, the protein levels of γ and δ were decreased to 30% and 10% of control levels, respectively. The content of the assembled ATP synthase decreased in accordance with the levels of the silenced subunits, which was also the case for most structural subunits. In contrast, the hydrophobic c subunit was increased to 130% or 180%, respectively and most of it was detected as aggregates of 150-400 kDa by 2D PAGE. In addition the IF1 protein was upregulated to 195% and 300% of control levels. Both γ and δ subunits silenced cells displayed decreased ATP synthase function - lowered rate of ADP-stimulated respiration, a two-fold increased sensitivity of respiration to inhibitor oligomycin, and impaired utilization of mitochondrial membrane potential for ADP phosphorylation. In summary, similar phenotype of γ, δ and ε subunit deficiencies suggest uniform requirement for assembled central stalk as driver of the c-oligomer attachment in the assembly process of mammalian ATP synthase.

• MeSH
• genový knockdown MeSH
• HEK293 buňky MeSH
• lidé MeSH
• mitochondriální proteiny genetika   metabolismus   MeSH
• mitochondrie genetika   metabolismus   MeSH
• protonové ATPasy genetika   metabolismus   MeSH
• spotřeba kyslíku fyziologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

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