Oxidation (β-like) of branched-chain keto acids (BCKAs) α-ketoisocaproate (KIC), α-ketoisovalerate (KIV), and α-ketomethylvalerate (KMV), yields FADH2, NADH (in order of KIC < KMV < KIV), and acetyl-CoA (KIC, KMV) or succinyl-CoA (KIV, KMV). Here, we examined whether BCKA-oxidation contributes to H2O2-dependent redox signaling, studied mechanism(s) of its generation, and investigated whether such H2O2 signal is required for BCKA-stimulated insulin secretion (BCKA-SIS) in pancreatic β-cells and islets. Using Amplex UltraRed, we detected BCKA-induced H2O2 release to the exterior of INS-1E cells and pancreatic islets (PIs) upon BCKA-SIS. This H2O2 signal determined closure of ATP-sensitive K+ channels and enabled Ca2+oscillations. It was inhibited by the mitochondrial antioxidant SkQ1; partially by S1QEL, S3QEL (Complex I, III) superoxide-suppressors and by 80-90% after silencing of electron-transfer flavoprotein (ETF) ubiquinone (Q) oxidoreductase (ETFQOR). The H2O2 (redox) signal is generated i) due to the excessive ETFQOR QH2 input, which retards respiratory chain electron transport, providing surplus superoxide at Complex I site IQ (i.e., reversed electron transfer, representing an effective product inhibition of the Complex I QH2 output) and ii) due to the excessive incoming QH2 to the Complex III site IIIQo (minimum for KIV). 13C-incorporation from U-13C-KIC/KIV into various metabolites confirmed β-like oxidation and characterized auxiliary reactions. A causal dependence of BCKA-SIS in PIs on H2O2 generation was found at both phases, evidenced by non-constant correlations of insulin release vs. H2O2 release rates, similarly to glucose-stimulated insulin secretion. Thus, BCKA-stimulated insulin secretion requires coordinated peri-plasma-membrane elevations of ATP/ADP and H2O2, both arising from mitochondrial BCKA β-like oxidation.

Combining luminescence with magnetic resonance imaging (MRI) is a noninvasive approach that significantly improves detection sensitivity and diagnostic precision for severe diseases. In diabetes care, this approach considerably broadens the possibilities for monitoring of Langerhans islet transplantation by improving their detectability and quantification within existing imaging modalities, including MRI. To realize this concept, upconverting nanoparticles (UCNPs) appear particularly promising, providing bimodal MRI and luminescence with the ability of near-infrared (NIR) light to penetrate deep into tissues. In this work, novel monodisperse dumbbell-shaped core-shell UCNPs (CS-UCNPs) coated with poly(methyl vinyl ether-alt-maleic acid) (PMVEMA) are developed for the bimodal imaging of Langerhans islets. Codoping of Fe, Yb, and Er ions in the NaYF4 host matrix, along with the presence of NaGdF4:Nd, Yb, Tb shell, increases both r 1 and r 2 relaxivities and upconversion luminescence in the red region, which is suitable for in vivo applications. The biocompatible PMVEMA coating ensures colloidal stability of the particles in aqueous physiological fluids and their nontoxicity. The potential of CS-UCNPs for simultaneous MRI and optical visualization is tested on isolated Langerhans islets. The efficiency of in vivo visualization of CS-UCNP@PMVEMA-labeled Langerhans islets transplanted under the kidney capsule in a rat model is investigated using T 1-, T 2-, and T 2*-weighted MRI sequences.

Abstract 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 abstract

In the diagnostics of diabetes, specific targeting of drugs (e.g., liraglutide) to insulin-deficient β-cells with their simultaneous noninvasive imaging is currently needed. In this report, liraglutide (LGL)-conjugated poly(methyl vinyl ether-alt-maleic acid) (PMVEMA)-coated core-shell NaYF4:Yb,Er,Fe@NaYF4:Nd upconversion nanoparticles (CS-UCNPs) have been developed, thoroughly physicochemically characterized, and evaluated in vivo. Novel codoping of Fe2+, Yb3+, and Er3+ ions in the host NaYF4 induced upconversion emission in the red region at both 980 and 808 nm excitation, making the particles suitable for deep-tissue imaging. Surface functionalization with PMVEMA provided colloidal stability and facilitated covalent conjugation with LGL, enabling targeted binding to GLP-1 receptors on pancreatic β-cells, increasing glucose-stimulated insulin secretion from isolated Langerhans islets. Biocompatibility of CS-UCNP@PMVEMA-LGL nanoparticles was confirmed by the trypan blue dye exclusion assay. When the fluorescent dye Flamma was conjugated to the nanoparticles, in vivo fluorescence imaging revealed significantly enhanced accumulation of CS-UCNP@PMVEMA-LGL-Flamma nanoparticles in the pancreas 24 h after intramuscular injection compared with intravenous administration, with luminescence intensity approximately doubled. The improved pancreatic targeting efficiency was attributed to enhanced binding to GLP-1 receptors. Confocal microscopy and elemental analysis confirmed receptor-mediated uptake of the nanoparticles by internalization and their localization within pancreatic β-cells. These findings highlight the potential of CS-UCNP@PMVEMA-LGL nanoparticles as biocompatible targetable imaging agents with future applications in pancreatic diagnostics.

Significance: Type 2 diabetes as a world-wide epidemic is characterized by the insulin resistance concomitant to a gradual impairment of β-cell mass and function (prominently declining insulin secretion) with dysregulated fatty acids (FAs) and lipids, all involved in multiple pathological development. Recent Advances: Recently, redox signaling was recognized to be essential for insulin secretion stimulated with glucose (GSIS), branched-chain keto-acids, and FAs. FA-stimulated insulin secretion (FASIS) is a normal physiological event upon postprandial incoming chylomicrons. This contrasts with the frequent lipotoxicity observed in rodents. Critical Issues: Overfeeding causes FASIS to overlap with GSIS providing repeating hyperinsulinemia, initiates prediabetic states by lipotoxic effects and low-grade inflammation. In contrast the protective effects of lipid droplets in human β-cells counteract excessive lipids. Insulin by FASIS allows FATP1 recruitment into adipocyte plasma membranes when postprandial chylomicrons come late at already low glycemia. Future Directions: Impaired states of pancreatic β-cells and peripheral organs at prediabetes and type 2 diabetes should be revealed, including the inter-organ crosstalk by extracellular vesicles. Details of FA/lipid molecular physiology are yet to be uncovered, such as complex phenomena of FA uptake into cells, postabsorptive inactivity of G-protein-coupled receptor 40, carnitine carrier substrate specificity, the role of carnitine-O-acetyltransferase in β-cells, and lipid droplet interactions with mitochondria. Antioxid. Redox Signal. 42, 566-622.

We describe a new concept for preparation of ultrasmall NaYF4:Yb,Er upconversion nanoparticles (UCNPs) with a diameter of 7 nm, depending on the amount of water added in the polymerization mixture, which affects the nucleation and growth of the particles. The morphology and structure of the nanoparticles were thoroughly characterized both in the dried state (TEM including elemental analysis and electron diffraction) and in solution (small and wide-angle X-ray scattering and dynamic light scattering). A thick NaYF4 shell was subsequently introduced onto the particles, which significantly increased the luminescence by minimizing surface quenching effects and passivating the core from the surrounding environment. To make the particles dispersible in the aqueous environment natural for biological applications, they were coated with a similar to 6 nm thick hydrophilic silica layer. This increased the size of core and core-shell UCNPs to 20 and similar to 50 nm. All the developed particles exhibited non-cytotoxicity tested in insulinoma INS-1E cells. The upconversion luminescence of these nanoparticles incubated with INS-1E cells showed a similar pattern to that of the particles themselves. The small biocompatible UCNPs developed in this study are promising candidates for non-invasive and non-destructive applications in bioimaging. Thanks to their advantageous properties, i.e., small size, adjustable optical properties and ability to interact with and easily penetrate cells, they are suitable for future use in platforms for targeted drug delivery and advanced diagnostic technologies.

We asked whether acute redox signaling from mitochondria exists concomitantly to fatty acid- (FA-) stimulated insulin secretion (FASIS) at low glucose by pancreatic β-cells. We show that FA β-oxidation produces superoxide/H2O2, providing: i) mitochondria-to-plasma-membrane redox signaling, closing KATP-channels synergically with elevated ATP (substituting NADPH-oxidase-4-mediated H2O2-signaling upon glucose-stimulated insulin secretion); ii) activation of redox-sensitive phospholipase iPLA2γ/PNPLA8, cleaving mitochondrial FAs, enabling metabotropic GPR40 receptors to amplify insulin secretion (IS). At fasting glucose, palmitic acid stimulated IS in wt mice; palmitic, stearic, lauric, oleic, linoleic, and hexanoic acids also in perifused pancreatic islets (PIs), with suppressed 1st phases in iPLA2γ/PNPLA8-knockout mice/PIs. Extracellular/cytosolic H2O2-monitoring indicated knockout-independent redox signals, blocked by mitochondrial antioxidant SkQ1, etomoxir, CPT1 silencing, and catalase overexpression, all inhibiting FASIS, keeping ATP-sensitive K+-channels open, and diminishing cytosolic [Ca2+]-oscillations. FASIS in mice was a postprandially delayed physiological event. Redox signals of FA β-oxidation are thus documented, reaching the plasma membrane, essentially co-stimulating IS.

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.

ObjectiveBy exposing mice carrying a deletion of NADPH oxidase isoform 4, NOX4, specifically in pancreatic beta cells (beta NOX4-/-) to nutrient excess stimulated by a high-fat diet (HFD), this study aimed to elucidate the role of beta-cell redox status in the development of meta-inflammation within the diabetic phenotype.MethodsThe authors performed basic phenotyping of beta NOX4-/- mice on HFD involving insulin and glycemic analyses, histochemistry of adipocytes, indirect calorimetry, and cytokine analyses. To characterize local inflammation, the study used caspase-1 activity assay, interleukin-1 beta immunochemistry, and real-time polymerase chain reaction during coculturing of beta cells with macrophages.ResultsThe phenotype of beta NOX4-/- mice on HFD was not associated with hyperinsulinemia and hyperglycemia but showed accumulation of excessive lipids in epididymal fat and beta cells. Surprisingly, mice showed significantly reduced systemic inflammation. Decreased interleukin-1 beta protein levels and downregulated NLRP3-inflammasome activity were observed on chronic glucose overload in beta NOX4-/- isolated islets and NOX4-silenced INS1-E cells resulting in attenuated proinflammatory polarization of macrophages/monocytes in vitro and in situ and reduced local islet inflammation.ConclusionsExperimental evidence suggests that NOX4 pro-oxidant activity in beta cells is involved in NLRP3-inflammasome activation during chronic nutrient overload and participates in local inflammatory signaling and perhaps toward peripheral tissues, contributing to a diabetic inflammatory phenotype. Redox signaling is involved in insulin secretion on glucose induction in pancreatic beta cells (NADPH oxidase isoform 4 [NOX4] is the major player), whereas oxidative stress in beta cells leads to the development of type 2 diabetes. Sustained activation of NOX4 in beta cells by chronic overnutrition induces the inflammasome and IL-1 beta production, leading to macrophage activation and local inflammation. Thus, beta NOX4-/- mice do not develop chronic inflammation on HFD, suggesting the role of sustained NOX4 activation in diabetes development. Redox regulation in beta cells is an important player in their physiology and NOX4 might be a potential pharmacological target during chronic nutritional overload.image