Andrea Dlasková

The endoplasmic reticulum (ER) and mitochondria maintain a dynamic structural partnership essential for pancreatic β-cell homeostasis, yet the high-resolution 3D remodeling of these networks under stress conditions remains poorly defined. We employed Focused Ion Beam Scanning Electron Microscopy (FIB-SEM) to perform 3D reconstructions of INS1E cells subjected to mitochondrial respiratory chain inhibition, uncoupling, and exogenous oxidative stress. Quantitative analysis revealed that mitochondrial dysfunction induces profound ultrastructural transitions, characterized by significant luminal swelling of the ER, expansion of the perinuclear space, and mitochondrial diameter enlargement. 3D volume imaging identified a coordinated fragmentation of both ER and mitochondrial networks into discrete, spatially separated structures—a phenomenon distinct from the reticular morphology observed in control cells. The similarity between respiratory inhibition- and H ₂ O ₂ -induced phenotypes, together with preservation of ER structure following mitochondrial uncoupling, suggests a potential contribution of reactive oxygen species to the observed remodeling process. Despite this extensive organelle breakdown, interorganelle membrane contact sites were not only preserved but expanded under stress conditions. We further provide a quantitative description of nuclear envelope–mitochondria contact sites (NAMs), demonstrating their selective remodeling during mitochondrial dysfunction. Our findings provide a high-resolution structural framework for organelle remodeling in β-cells, demonstrating that membrane contact sites are actively preserved and reorganized despite profound organelle fragmentation. Graphical

The endoplasmic reticulum (ER) and mitochondria maintain a dynamic structural partnership essential for pancreatic β-cell homeostasis, yet the high-resolution 3D remodeling of these networks under stress conditions remains poorly defined.We employed Focused Ion Beam Scanning Electron Microscopy (FIB-SEM) to perform 3D reconstructions of INS1E cells subjected to mitochondrial respiratory chain inhibition, uncoupling, and exogenous oxidative stress. Quantitative analysis revealed that mitochondrial dysfunction induces profound ultrastructural transitions, characterized by significant luminal swelling of the ER, expansion of the perinuclear space, and mitochondrial diameter enlargement. 3D volume imaging identified a coordinated fragmentation of both ER and mitochondrial networks into discrete, spatially separated structures—a phenomenon distinct from the reticular morphology observed in control cells. The similarity between respiratory inhibition- and H2O2-induced phenotypes, together with preservation of ER structure following mitochondrial uncoupling, suggests a potential contribution of reactive oxygen species to the observed remodeling process.Despite this extensive organelle breakdown, interorganelle membrane contact sites were not only preserved but expanded under stress conditions. We further provide a quantitative description of nuclear envelope–mitochondria contact sites (NAMs), demonstrating their selective remodeling during mitochondrial dysfunction. Our findings provide a high-resolution structural framework for organelle remodeling in β-cells, demonstrating that membrane contact sites are actively preserved and reorganized despite profound organelle fragmentation.

Significance: Mitochondrial (mt) reticulum network in the cell possesses amazing ultramorphology of parallel lamellar cristae, formed by the invaginated inner mitochondrial membrane. Its non-invaginated part, the inner boundary membrane (IBM) forms a cylindrical sandwich with the outer mitochondrial membrane (OMM). Crista membranes (CMs) meet IBM at crista junctions (CJs) of mt cristae organizing system (MICOS) complexes connected to OMM sorting and assembly machinery (SAM). Cristae dimensions, shape, and CJs have characteristic patterns for different metabolic regimes, physiological and pathological situations. Recent Advances: Cristae-shaping proteins were characterized, namely rows of ATP-synthase dimers forming the crista lamella edges, MICOS subunits, optic atrophy 1 (OPA1) isoforms and mitochondrial genome maintenance 1 (MGM1) filaments, prohibitins, and others. Detailed cristae ultramorphology changes were imaged by focused-ion beam/scanning electron microscopy. Dynamics of crista lamellae and mobile CJs were demonstrated by nanoscopy in living cells. With tBID-induced apoptosis a single entirely fused cristae reticulum was observed in a mitochondrial spheroid. Critical Issues: The mobility and composition of MICOS, OPA1, and ATP-synthase dimeric rows regulated by post-translational modifications might be exclusively responsible for cristae morphology changes, but ion fluxes across CM and resulting osmotic forces might be also involved. Inevitably, cristae ultramorphology should reflect also mitochondrial redox homeostasis, but details are unknown. Disordered cristae typically reflect higher superoxide formation. Future Directions: To link redox homeostasis to cristae ultramorphology and define markers, recent progress will help in uncovering mechanisms involved in proton-coupled electron transfer via the respiratory chain and in regulation of cristae architecture, leading to structural determination of superoxide formation sites and cristae ultramorphology changes in diseases. Antioxid. Redox Signal. 39, 635–683.

Previously, a number of ~ 1.4 of mitochondrial DNA (mtDNA) molecules in a single nucleoid was reported, which would reflect a minimum nucleoid division. We applied 3D-double-color direct stochastic optical reconstruction microscopy (dSTORM), i.e. nanoscopy with ~ 25–40 nm x,y-resolution, together with our novel method of Delaunay segmentation of 3D data to identify unbiased 3D-overlaps. Noncoding D-loops were recognized in HeLa cells by mtDNA fluorescence in situ hybridization (mtFISH) 7S-DNA 250-bp probe, containing biotin, visualized by anti-biotin/Cy3B-conjugated antibodies. Other mtFISH probes with biotin or Alexa Fluor 647 (A647) against ATP6-COX3 gene overlaps (1,100 bp) were also used. Nucleoids were imaged by anti-DNA/(A647-)-Cy3B-conjugated antibodies. Resulting histograms counting mtFISH-loci/nucleoid overlaps demonstrated that 45% to 70% of visualized nucleoids contained two or more D-loops or ATP6-COX3-loci, indicating two or more mtDNA molecules per nucleoid. With increasing number of mtDNA per nucleoid, diameters were larger and their distribution histograms peaked at ~ 300 nm. A wide nucleoid diameter distribution was obtained also using 2D-STED for their imaging by anti-DNA/A647. At unchanged mtDNA copy number in osteosarcoma 143B cells, TFAM expression increased nucleoid spatial density 1.67-fold, indicating expansion of existing mtDNA and its redistribution into more nucleoids upon the higher TFAM/mtDNA stoichiometry. Validation of nucleoid imaging was also done with two TFAM mutants unable to bend or dimerize, respectively, which reduced both copy number and nucleoid spatial density by 80%. We conclude that frequently more than one mtDNA molecule exists within a single nucleoid in HeLa cells and that mitochondrial nucleoids do exist in a non-uniform size range.

In this review, we place redox signaling in pancreatic β-cells to the context with signaling pathways leading to insulin secretion, acting for example upon the action of incretins (GLP-1, GIP) and the metabotropic receptor GPR40. Besides a brief description of ion channel participation in depolarization/repolarization of the plasma membrane, we emphasize a prominent role of the elevated glucose level in pancreatic β-cells during glucose-stimulated insulin secretion (GSIS). We focus on our recent findings, which revealed that for GSIS, not only elevated ATP synthesis is required, but also fundamental redox signaling originating from the NADPH oxidase 4- (NOX4-) mediated H2O2 production. We hypothesized that the closing of the ATP-sensitive K+ channel (KATP) is only possible when both ATP plus H2O2 are elevated in INS-1E cells. KATP alone or with synergic channels provides an element of logical sum, integrating both metabolic plus redox homeostasis. This is also valid for other secretagogues, such as branched chain ketoacids (BCKAs); and partly for fatty acids (FAs). Branched chain aminoacids, leucine, valine and isoleucine, after being converted to BCKAs are metabolized by a series of reactions resembling β-oxidation of FAs. This increases superoxide formation in mitochondria, including its portion elevated due to the function of electron transfer flavoprotein ubiquinone oxidoreductase (ETF:QOR). After superoxide conversion to H2O2 the oxidation of BCKAs provides the mitochondrial redox signaling extending up to the plasma membrane to induce its depolarization together with the elevated ATP. In contrast, experimental FA-stimulated insulin secretion in the presence of non-stimulating glucose concentrations is predominantly mediated by GPR40, for which intramitochondrial redox signaling activates phospholipase iPLA2γ, cleaving free FAs from mitochondrial membranes, which diffuse to the plasma membrane and largely amplify the GPR40 response. These events are concomitant to the insulin release due to the metabolic component. Hypothetically, redox signaling may proceed by simple H2O2 diffusion or via an SH-relay enabled by peroxiredoxins to target proteins. However, these aspects have yet to be elucidated.

Pancreatic β-cell insulin secretion, which responds to various secretagogues and hormonal regulations, is reviewed here, emphasizing the fundamental redox signaling by NADPH oxidase 4- (NOX4-) mediated H₂O₂ production for glucose-stimulated insulin secretion (GSIS). There is a logical summation that integrates both metabolic plus redox homeostasis because the ATP-sensitive K⁺ channel (K_ATP) can only be closed when both ATP and H₂O₂ are elevated. Otherwise ATP would block K_ATP, while H₂O₂ would activate any of the redox-sensitive nonspecific calcium channels (NSCCs), such as TRPM2. Notably, a 100%-closed K_ATP ensemble is insufficient to reach the −50 mV threshold plasma membrane depolarization required for the activation of voltage-dependent Ca²⁺ channels. Open synergic NSCCs or Cl⁻ channels have to act simultaneously to reach this threshold. The resulting intermittent cytosolic Ca²⁺-increases lead to the pulsatile exocytosis of insulin granule vesicles (IGVs). The incretin (e.g., GLP-1) amplification of GSIS stems from receptor signaling leading to activating the phosphorylation of TRPM channels and effects on other channels to intensify integral Ca²⁺-influx (fortified by endoplasmic reticulum Ca²⁺). ATP plus H₂O₂ are also required for branched-chain ketoacids (BCKAs); and partly for fatty acids (FAs) to secrete insulin, while BCKA or FA β-oxidation provide redox signaling from mitochondria, which proceeds by H₂O₂ diffusion or hypothetical SH relay via peroxiredoxin “redox kiss” to target proteins.

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 m M ; pancreatic islets from 5 m M ) to 25 m M glucose decreased matrix superoxide release rates (MitoSOX Red monitoring validated by MitoB) and H ₂ O ₂ (mitoHyPer, subtracting mitoSypHer emission). Novel double-channel fluorescence lifetime imaging, approximating free mitochondrial matrix NADH _F, 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 NADPH _F (iNAP1 fluorescence monitoring) at the expense of matrix NADH _F , was indicated, using citrate (2-oxoglutarate) carrier inhibitors and cytosolic malic enzyme silencing: All changes vanished on these manipulations. ¹³ C-incorporation from ¹³ C-L-glutamine into ¹³ C-citrate reflected the pyruvate/isocitrate shuttle. Matrix NADPH _F (iNAP3 monitored) decreased. With decreasing glucose, the suppressor of Complex III site Q electron leak (S3QEL) suppressor caused a higher Complex I I _F site contribution, but a lower superoxide fraction ascribed to the Complex III site III _Qo . Thus, the diminished matrix NADH _F /NAD ⁺ _F decreased Complex I flavin site I _F 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 NADH _F /NAD ⁺ _F (NADPH _F /NADP ⁺ _F ) at increasing cytosolic NADPH _F 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.

Transcript levels for selected ATP synthase membrane F_O-subunits—including DAPIT—in INS-1E cells were found to be sensitive to lowering glucose down from 11 mM, in which these cells are routinely cultured. Depending on conditions, the diminished mRNA levels recovered when glucose was restored to 11 mM; or were elevated during further 120 min incubations with 20-mM glucose. Asking whether DAPIT expression may be elevated by hyperglycemia in vivo, we studied mice with hyaluronic acid implants delivering glucose for up to 14 days. Such continuous two-week glucose stimulations in mice increased DAPIT mRNA by >5-fold in isolated pancreatic islets (ATP synthase F₁α mRNA by 1.5-fold). In INS-1E cells, the glucose-induced ATP increment vanished with DAPIT silencing (6% of ATP rise), likewise a portion of the mtDNA-copy number increment. With 20 and 11-mM glucose the phosphorylating/non-phosphorylating respiration rate ratio diminished to ~70% and 96%, respectively, upon DAPIT silencing, whereas net GSIS rates accounted for 80% and 90% in USMG5/DAPIT-deficient cells. Consequently, the sufficient DAPIT expression and complete ATP synthase assembly is required for maximum ATP synthesis and mitochondrial biogenesis, but not for insulin secretion as such. Elevated DAPIT expression at high glucose further increases the ATP synthesis efficiency.

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.