Mitochondrial Network Morphology Research Articles

AntagomiR-103 and -107 Treatment Affects Cardiac Function and Metabolism

MicroRNA-103/107 regulate systemic glucose metabolism and insulin sensitivity. For this reason, inhibitory strategies for these microRNAs are currently being tested in clinical trials. Given the high metabolic demands of the heart and the abundant cardiac expression of miR-103/107, we questioned whether antagomiR-mediated inhibition of miR-103/107 in C57BL/6J mice impacts on cardiac function. Notably, fractional shortening decreased after 6 weeks of antagomiR-103 and -107 treatment. This was paralleled by a prolonged systolic radial and circumferential time to peak and by a decreased global strain rate. Histology and electron microscopy showed reduced cardiomyocyte area and decreased mitochondrial volume and mitochondrial cristae density following antagomiR-103 and -107. In line, antagomiR-103 and -107 treatment decreased mitochondrial OXPHOS complexes’ protein levels compared to scrambled, as assessed by mass spectrometry-based label-free quantitative proteomics. MiR-103/107 inhibition in primary cardiomyocytes did not affect glycolysis rates, but it decreased mitochondrial reserve capacity, reduced mitochondrial membrane potential, and altered mitochondrial network morphology, as assessed by live-cell imaging. Our data indicate that antagomiR-103 and -107 decrease cardiac function, cardiomyocyte size, and mitochondrial oxidative capacity in the absence of pathological stimuli. These data raise concern about the possible cardiac implications of the systemic use of antagomiR-103 and -107 in the clinical setting, and careful cardiac phenotyping within ongoing trials is highly recommended.

Absorption of blue light by cigarette smoke components is highly toxic for retinal pigmented epithelial cells.

Lesion to the retinal pigment epithelium (RPE) is a crucial event in the development of age-related macular degeneration (AMD), the leading cause of blindness in industrialized countries. Tobacco smoking and high-energy visible blue (HEV; 400-500nm) light exposure are major environmental risk factors for AMD. Individually, they have been shown to cause damage to the RPE. Tobacco smoke contains toxic polycyclic aromatic hydrocarbons (PAH) that can accumulate in RPE and which absorb HEV light. It can thus be postulated that the interaction between both factors in RPE cells can have a synergic toxic effect to the RPE. To test this hypothesis, cultured human RPE cells (ARPE19) were treated with nanomolar concentrations of benzo[a]pyrene (BaP) or indeno[1,2,3-cd]pyrene (IcdP), then exposed to HEV light using an irradiation system that mimics the solar spectrum normally transmitted to the retina through the human ocular media. Using mitochondrial network morphology changes and key features of AMD-related RPE defects such as apoptotic cell death and oxidative stress, we demonstrate that a synergistic phototoxicity is generated when nanomolar concentrations (≤ 500nM) of IcdP interact with sub-lethal amounts of HEV light. Indeed, we found IcdP to be at least 3000 times more toxic for RPE cells when irradiated with HEV light. This synergy translates into disruption of mitochondrial network, ROS enhanced accumulation and apoptosis of RPE cells. Our results underline an important interplay between two environmental risk factors involved in AMD progression and strongly indicate that IcdP, upon interaction with HEV light, may initiate the biological mechanisms underlying the association between cigarette smoking and AMD-related RPE degeneration.

85 - Heme biosynthesis and trafficking

Heme is an essential cofactor involved in a plethora of vital functions. Heme is required to be in most subcellular compartments, but the way, in which it is transported from the mitochondria - its site of synthesis - to those compartments is poorly understood. Utilizing ratiometric, fluorescent heme sensors, we found that the GTPases in control of mitochondrial fusion, Mgm1 and fission, Dnm1, are positive and negative regulators of heme trafficking to the nucleus, respectively. Furthermore, we discovered that mitochondrial network morphology is under the control of heme biosynthesis. Our results indicate that mitochondrial dynamics and heme trafficking are integrally coupled.

Mice harbouring a SCA28 patient mutation in AFG3L2 develop late-onset ataxia associated with enhanced mitochondrial proteotoxicity

Spinocerebellar ataxia 28 is an autosomal dominant neurodegenerative disorder caused by missense mutations affecting the proteolytic domain of AFG3L2, a major component of the mitochondrial m-AAA protease. However, little is known of the underlying pathogenetic mechanisms or how to treat patients with SCA28. Currently available Afg3l2 mutant mice harbour deletions that lead to severe, early-onset neurological phenotypes that do not faithfully reproduce the late-onset and slowly progressing SCA28 phenotype. Here we describe production and detailed analysis of a new knock-in murine model harbouring an Afg3l2 allele carrying the p.Met665Arg patient-derived mutation. Heterozygous mutant mice developed normally but adult mice showed signs of cerebellar ataxia detectable by beam test. Although cerebellar pathology was negative, electrophysiological analysis showed a trend towards increased spontaneous firing in Purkinje cells from heterozygous mutants with respect to wild-type controls. As homozygous mutants died perinatally with evidence of cardiac atrophy, for each genotype we generated mouse embryonic fibroblasts (MEFs) to investigate mitochondrial function. MEFs from mutant mice showed altered mitochondrial bioenergetics, with decreased basal oxygen consumption rate, ATP synthesis and mitochondrial membrane potential. Mitochondrial network formation and morphology was altered, with greatly reduced expression of fusogenic Opa1 isoforms. Mitochondrial alterations were also detected in cerebella of 18-month-old heterozygous mutants and may be a hallmark of disease. Pharmacological inhibition of de novo mitochondrial protein translation with chloramphenicol caused reversal of mitochondrial morphology in homozygous mutant MEFs, supporting the relevance of mitochondrial proteotoxicity for SCA28 pathogenesis and therapy development.

Mitochondria reorganization upon proliferation arrest predicts individual yeast cell fate.

Most cells spend the majority of their life in a non-proliferating state. When proliferation cessation is irreversible, cells are senescent. By contrast, if the arrest is only temporary, cells are defined as quiescent. These cellular states are hardly distinguishable without triggering proliferation resumption, hampering thus the study of quiescent cells properties. Here we show that quiescent and senescent yeast cells are recognizable based on their mitochondrial network morphology. Indeed, while quiescent yeast cells display numerous small vesicular mitochondria, senescent cells exhibit few globular mitochondria. This allowed us to reconsider at the individual-cell level, properties previously attributed to quiescent cells using population-based approaches. We demonstrate that cell's propensity to enter quiescence is not influenced by replicative age, volume or density. Overall, our findings reveal that quiescent cells are not all identical but that their ability to survive is significantly improved when they exhibit the specific reorganization of several cellular machineries.

AMPK activation negatively regulates GDAP1, which influences metabolic processes and circadian gene expression in skeletal muscle

ObjectiveWe sought to identify AMPK-regulated genes via bioinformatic analysis of microarray data generated from skeletal muscle of animal models with genetically altered AMPK activity. We hypothesized that such genes would play a role in metabolism. Ganglioside-induced differentiation-associated protein 1 (GDAP1), a gene which plays a role in mitochondrial fission and peroxisomal function in neuronal cells but whose function in skeletal muscle is undescribed, was identified and further validated. AMPK activation reduced GDAP1 expression in skeletal muscle. GDAP1 expression was elevated in skeletal muscle from type 2 diabetic patients but decreased after acute exercise. MethodsThe metabolic impact of GDAP1 silencing was determined in primary skeletal muscle cells via siRNA-transfections. Confocal microscopy was used to visualize whether silencing GDAP1 impacted mitochondrial network morphology and membrane potential. ResultsGDAP1 silencing increased mitochondrial protein abundance, decreased palmitate oxidation, and decreased non-mitochondrial respiration. Mitochondrial morphology was unaltered by GDAP1 silencing. GDAP1 silencing and treatment of cells with AMPK agonists altered several genes in the core molecular clock machinery. ConclusionWe describe a role for GDAP1 in regulating mitochondrial proteins, circadian genes, and metabolic flux in skeletal muscle. Collectively, our results implicate GDAP1 in the circadian control of metabolism.

A Multifunctional Chemical Probe for the Measurement of Local Micropolarity and Microviscosity in Mitochondria.

The measurement of physicochemical parameters in living cells can provide information on individual cellular organelles, helping us to understand subcellular function in health and disease. While organelle-specific chemical probes have allowed qualitative evaluation of microenvironmental variations, the simultaneous quantification of mitochondrial local microviscosity (ηm ) and micropolarity (ϵm ), along with concurrent structural variations, has remained an unmet need. Herein, we describe a new multifunctional mitochondrial probe (MMP) for simultaneous monitoring of ηm and ϵm by fluorescence lifetime and emission intensity recordings, respectively. The MMP enables highly precise measurements of ηm and ϵm in the presence of a variety of agents perturbing cellular function, and the observed changes can also be correlated with alterations in mitochondrial network morphology and motility. This strategy represents a promising tool for the analysis of subtle changes in organellar structure.

Ectromelia Virus Affects Mitochondrial Network Morphology, Distribution, and Physiology in Murine Fibroblasts and Macrophage Cell Line.

Mitochondria are multifunctional organelles that participate in numerous processes in response to viral infection, but they are also a target for viruses. The aim of this study was to define subcellular events leading to alterations in mitochondrial morphology and function during infection with ectromelia virus (ECTV). We used two different cell lines and a combination of immunofluorescence techniques, confocal and electron microscopy, and flow cytometry to address subcellular changes following infection. Early in infection of L929 fibroblasts and RAW 264.7 macrophages, mitochondria gathered around viral factories. Later, the mitochondrial network became fragmented, forming punctate mitochondria that co-localized with the progeny virions. ECTV-co-localized mitochondria associated with the cytoskeleton components. Mitochondrial membrane potential, mitochondrial fission–fusion, mitochondrial mass, and generation of reactive oxygen species (ROS) were severely altered later in ECTV infection leading to damage of mitochondria. These results suggest an important role of mitochondria in supplying energy for virus replication and morphogenesis. Presumably, mitochondria participate in transport of viral particles inside and outside of the cell and/or they are a source of membranes for viral envelope formation. We speculate that the observed changes in the mitochondrial network organization and physiology in ECTV-infected cells provide suitable conditions for viral replication and morphogenesis.

Metabolic Reprogramming in Amyotrophic Lateral Sclerosis

Mitochondrial dysfunction in the spinal cord is a hallmark of amyotrophic lateral sclerosis (ALS), but the neurometabolic alterations during early stages of the disease remain unknown. Here, we investigated the bioenergetic and proteomic changes in ALS mouse motor neurons and patients’ skin fibroblasts. We first observed that SODG93A mice presymptomatic motor neurons display alterations in the coupling efficiency of oxidative phosphorylation, along with fragmentation of the mitochondrial network. The proteome of presymptomatic ALS mice motor neurons also revealed a peculiar metabolic signature with upregulation of most energy-transducing enzymes, including the fatty acid oxidation (FAO) and the ketogenic components HADHA and ACAT2, respectively. Accordingly, FAO inhibition altered cell viability specifically in ALS mice motor neurons, while uncoupling protein 2 (UCP2) inhibition recovered cellular ATP levels and mitochondrial network morphology. These findings suggest a novel hypothesis of ALS bioenergetics linking FAO and UCP2. Lastly, we provide a unique set of data comparing the molecular alterations found in human ALS patients’ skin fibroblasts and SODG93A mouse motor neurons, revealing conserved changes in protein translation, folding and assembly, tRNA aminoacylation and cell adhesion processes.

Cardiomyocyte mitochondrial dynamics in health and disease and the role of exercise training: A brief review

Mitochondria as dynamic organelle constantly undergo fusion and fission reactions, leading to continuous reconstruction of the mitochondrial network for elongated or fragmentation shapes and ultimately the mitophagy. This mitochondrial network dynamics is sensitive to stress and different physiological conditions and plays an essential role in cell function and survival during pathophysiological conditions. There is a strong interaction between the mitochondrial network morphology and proteins involved in energy metabolism and dynamics. It is suggested that changes in cellular energy status during exercise training are due to mitochondrial network dynamics and mitophagy. Accordingly, growing evidence reveals that exercise training results in alterations in mitochondrial phenotype and dynamics that resist apoptotic stimuli and ischemia-reperfusion-induced mitochondrial damage. However, the signaling pathways of mitochondrial dynamics and mitophagy regulation during exercise training are still interesting areas of research. In this review, we focus on the recent findings addressing cellular signaling mechanisms of mitochondrial dynamics and cardiac mitophagy in response to exercise training and the pathological stimulus in heart disorders.

204 - Neuroglobin Overexpression Promotes Mitochondrial Fusion and Prevents Mitochondrial Network Fragmentation in SHSY5Y Cells Subjected to Pro-apoptotic Conditions

Oxidative stress and hypoxia play a central role in neuronal injury and cell death in acute and chronic pathological conditions. Neuroglobin (NGB) is a monomeric heme-protein found in neurons that, when overexpressed, confers resistance to apoptosis cause by oxidative stress, hypoxia, and neurotoxicity. Although NGB has been traditionally considered a cytoplasmic protein, recent findings indicate that NGB can localize to mitochondria. However, we have an incomplete understanding of the details of NGB’s mitochondrial interactions and effects. Here, we investigated the effect of NGB overexpression on mitochondrial network morphology in SHSY5Y neuroblastoma cell line using a quantitative approach to measure mitochondrial network features including network size, branch length, and the overall abundance of mitochondria. We found that NGB overexpression caused the formation of larger and more highly branched mitochondrial networks and greater mitochondrial abundance. NGB overexpression also prevented mitochondrial fragmentation, an early event in the apoptotic pathway, in cells exposed to hypoxia, hypoxia/reperfusion, or hydrogen peroxide treatment. These data demonstrate interactions between NGB and mitochondrial fusion/fission processes. We are now exploring whether this effect is due to a direct interaction between mitochondria and Ngb, or involves cytosplasmic signaling pathways. Taken together, these observations have the potential to open new avenues to develop therapeutic strategies against neurodegenerative disease, where dysfunctional mitochondrial dynamics represent a common and prominent early pathological feature.

Estradiol and Estrogen Receptor-β Agonist Diarylpropionitrile Promote Mitochondrial Network fusion

Estrogens exert many effects on mitochondrial function via targeting both nuclear and mitochondrial encoded genes. Recent reports indicate that the expression of mitochondrial fusion and fission proteins might be downstream targets of estradiol (E2). However, there has been no quantitative evaluation of E2 effects on mitochondrial network characteristics. We used MiNA, the Mitochondrial Network Analysis tool recently developed in our laboratory, to quantitatively measure mitochondrial network features in cells treated with E2 or the selective estrogen receptor-α and β modulators (SERMs) propylpyrazole triol (PPT) and diarylpropionitrile (DPN), respectively. 48h treatment of mouse C2C12 myoblasts with various concentrations of either E2 or DPN, but not PPT, significantly modified mitochondrial network morphology. Both E2 and DPN approximately doubled the number of mitochondrial networks as well as the number of branches per network, while PPT had no effect on these parameters. Similarly, both E2 and DPN significantly increased the mitochondrial footprint, a measure of mitochondrial abundance, while PPT failed to modulate this feature. These data indicate that ER-β agonists strongly promote mitochondrial fusion. Thus, these observations provide initial quantitative evidence of beneficial actions of SERMs such as DPN as a pharmacological tool for targeting mitochondrial fusion in pathological conditions characterized by deficient mitochondrial network formation, such as certain neurodegenerative diseases and cancers.

211 - A Generalized Image Workflow for Quantitative Analysis of Mitochondrial Networks

Mitochondria undergo constant fusion, fission, and morphological change in response to environmental stressors and a cell's energy demands. The ability of a mitochondrial network to undergo such changes plays a vital role in maintaining cellular physiological homeostasis. Disruptions have been linked to cellular dysfunction, disease onset, and developmental abnormalities. To effectively investigate such processes, it is important to quantitatively describe structural features of the mitochondrial network. Methods by others have been developed for analysis of single images, high content images, and 3-dimensional stacks acquired through fluorescence microscopy. These efforts have provided a foundation for our development of a generalized, simple workflow using ImageJ, encapsulated into a plugin for convenience and portability. The plugin, called GeNA (Generalized Network Analysis) is an offshoot of the MiNA macro (Valente et al. 2017; Acta Histol). GeNA provides significant improvements on MiNA in terms of performance and accuracy, and it is flexible enough to be used with many different applications. Here, we demonstrate its usage for investigating mitochondrial networks in 2D, 3D, and time series data sets. The GeNA plugin pack provides a free cross-platform solution that is easy to understand and implement. It conveniently quantifies key mitochondrial network morphology features, such as the mitochondrial area or volume, the number of branches in network-like structures, and the lengths of rod-like features and network branches.

MitoNEET-dependent formation of intermitochondrial junctions

MitoNEET (mNEET) is a dimeric mitochondrial outer membrane protein implicated in many facets of human pathophysiology, notably diabetes and cancer, but its molecular function remains poorly characterized. In this study, we generated and analyzed mNEET KO cells and found that in these cells the mitochondrial network was disturbed. Analysis of 3D-EM reconstructions and of thin sections revealed that genetic inactivation of mNEET did not affect the size of mitochondria but that the frequency of intermitochondrial junctions was reduced. Loss of mNEET decreased cellular respiration, because of a reduction in the total cellular mitochondrial volume, suggesting that intermitochondrial contacts stabilize individual mitochondria. Reexpression of mNEET in mNEET KO cells restored the WT morphology of the mitochondrial network, and reexpression of a mutant mNEET resistant to oxidative stress increased in addition the resistance of the mitochondrial network to H2O2-induced fragmentation. Finally, overexpression of mNEET increased strongly intermitochondrial contacts and resulted in the clustering of mitochondria. Our results suggest that mNEET plays a specific role in the formation of intermitochondrial junctions and thus participates in the adaptation of cells to physiological changes and to the control of mitochondrial homeostasis.

OPA1 Isoforms in the Hierarchical Organization of Mitochondrial Functions

OPA1 is a GTPase that controls mitochondrial fusion, cristae integrity, and mtDNA maintenance. In humans, eight isoforms are expressed as combinations of long and short forms, but it is unclear whether OPA1 functions are associated with specific isoforms and/or domains. To address this, we expressed each of the eight isoforms or different constructs of isoform 1 in Opa1-/- MEFs. We observed that any isoform could restore cristae structure, mtDNA abundance, and energetic efficiency independently of mitochondrial network morphology. Long forms supported mitochondrial fusion; short forms were better able to restore energetic efficiency. The complete rescue of mitochondrial network morphology required a balance of long and short forms of at least two isoforms, as shown by combinatorial isoform silencing and co-expression experiments. Thus, multiple OPA1 isoforms are required for mitochondrial dynamics, while any single isoform can support all other functions. These findings will be useful in designing gene therapies for patients with OPA1 haploinsufficiency.

The Interplay between Oncogenic Signaling Networks and Mitochondrial Dynamics.

Mitochondria are dynamic organelles that alter their organization in response to a variety of cellular cues. Mitochondria are central in many biologic processes, such as cellular bioenergetics and apoptosis, and mitochondrial network morphology can contribute to those physiologic processes. Some of the biologic processes that are in part governed by mitochondria are also commonly deregulated in cancers. Furthermore, patient tumor samples from a variety of cancers have revealed that mitochondrial dynamics machinery may be deregulated in tumors. In this review, we will discuss how commonly mutated oncogenes and their downstream effector pathways regulate the mitochondrial dynamics machinery to promote changes in mitochondrial morphology as well as the physiologic consequences of altered mitochondrial morphology for tumorigenic growth.

Quantitative image based analysis of endocrine disruptor effects on mitochondria morphology-function in prostate cancer cells

Endocrine Disrupting Compounds (EDC) are found in many everyday products, like food packaging, food preservative or additive, pesticides residues, etc… Widely distributed throughout the environment and bioaccumulable in living organisms, persistent organic pollutants (POPs) are a specific class of EDC that accumulate in fat deposit. Some of them have been recognized as causing adverse effects on human’s health such as diabetes and cancer by mimicking hormone effects on metabolism. Cancer cells display high metabolic flexibility allowing them to grow in various cellular environments and favoring their proliferative and invasive capacities. Mitochondria are key players in this complex interplay as they produce ROS, generate energy and participate in nucleotide synthesis and in glutamine metabolism of cancer cells. Regarding the importance of hormones on prostate cancer risk and outcomes, we are developing multiple parameters in vitro assays conducted in a high-throughput screening format relevant for prostate cancer metabolism and aggressiveness. This screening method includes, inter alia a microscopy based analysis of mitochondria structure and function. We analyzed the effects of five EDCs (Aldrin, BDE28, TCDD, PCB153, PFOA) identified in the plasma of patients on two prostate cancer cell lines, 22RV1 (androgen-responsive) and DU145 (androgen-unresponsive). Each compound was tested in a dose dependent manner to determine its effects on total mitochondrial mass, mitochondrial membrane potential, ROS production, mitochondrial biogenesis and mitophagy. In addition, we performed an image based computational analysis of the mitochondrial network morphology and dynamics. This strategy allows us to extract some quantitative parameters on the mitochondrial network as fragmentation index, compactness, average volume, etc. When combined, morphological and functional parameters allow us to discriminate subtle perturbations of the mitochondrial structure-function induced by POPs in prostate cancer cells. We are confident that this multiparameter analysis strategy could represent a new perspective in identification and characterization of EDC based on their effects on cell metabolism (phenoscore) in order to estimate their potential risk on human health.

SLP-2 interacts with Parkin in mitochondria and prevents mitochondrial dysfunction in Parkin-deficient human iPSC-derived neurons and Drosophila.

Mutations in the Parkin gene (PARK2) have been linked to a recessive form of Parkinson's disease (PD) characterized by the loss of dopaminergic neurons in the substantia nigra. Deficiencies of mitochondrial respiratory chain complex I activity have been observed in the substantia nigra of PD patients, and loss of Parkin results in the reduction of complex I activity shown in various cell and animal models. Using co-immunoprecipitation and proximity ligation assays on endogenous proteins, we demonstrate that Parkin interacts with mitochondrial Stomatin-like protein 2 (SLP-2), which also binds the mitochondrial lipid cardiolipin and functions in the assembly of respiratory chain proteins. SH-SY5Y cells with a stable knockdown of Parkin or SLP-2, as well as induced pluripotent stem cell-derived neurons from Parkin mutation carriers, showed decreased complex I activity and altered mitochondrial network morphology. Importantly, induced expression of SLP-2 corrected for these mitochondrial alterations caused by reduced Parkin function in these cells. In-vivo Drosophila studies showed a genetic interaction of Parkin and SLP-2, and further, tissue-specific or global overexpression of SLP-2 transgenes rescued parkin mutant phenotypes, in particular loss of dopaminergic neurons, mitochondrial network structure, reduced ATP production, and flight and motor dysfunction. The physical and genetic interaction between Parkin and SLP-2 and the compensatory potential of SLP-2 suggest a functional epistatic relationship to Parkin and a protective role of SLP-2 in neurons. This finding places further emphasis on the significance of Parkin for the maintenance of mitochondrial function in neurons and provides a novel target for therapeutic strategies.

Resveratrol stimulates mitochondrial fusion by a mechanism requiring mitofusin-2

Resveratrol (RES) is a plant-derived stilbene associated with a wide range of health benefits. Mitochondria are a key downstream target of RES, and in some cell types RES promotes mitochondrial biogenesis, altered cellular redox status, and a shift toward oxidative metabolism. Mitochondria exist as a dynamic network that continually remodels via fusion and fission processes, and the extent of fusion is related to cellular redox status and metabolism. We investigated RES's effects on mitochondrial network morphology in several cell lines using a quantitative approach to measure the extent of network fusion. 48 h continuous treatment with 10–20 μM RES stimulated mitochondrial fusion in C2C12 myoblasts, PC3 cancer cells, and mouse embryonic fibroblasts stimulated significant increases in fusion in all instances, resulting in larger and more highly branched mitochondrial networks. Mitofusin-2 (Mfn2) is a key protein facilitating mitochondrial fusion, and its expression was also stimulated by RES. Using Mfn2-null cells we demonstrated that RES's effects on mitochondrial fusion, cellular respiration rates, and cell growth are all dependent upon the presence of Mfn2. Taken together, these results demonstrate that Mfn2 and mitochondrial fusion are affected by RES in ways that appear to relate to RES's known effects on cellular metabolism and growth.

413 - Mitochondrial Superoxide Generation and Morphology Changes upon Glucose-Stimulated Insulin Secretion (GSIS) in INS-1E Cells

Mitochondria play a key role in insulin secretion by pancreatic beta cells due to their control over the cellular metabolism and namely ATP generation. Previous studies have shown that glucose levels have direct impact on superoxide formation by the mitochondrial respiratory chain. In turn, mitochondrial superoxide has been described to induce remodeling of mitochondrial morphology. In this study we examined mitochondrial superoxide formation under various glucose concentrations in association with mitochondrial morphology. To monitor in situ surplus superoxide O2●– released to the mitochondrial matrix (Jm) we employed confocal fluorescence microscopy time scanning of MitoSOX Red [1]. To assess 3D mitochondrial network morphology we applied SIM (Structured Illumination Microscopy). We compared Jm rates in INS-1E cultivated for 2 hrs at 0 mM, 3 mM, 11 mM and 20 mM glucose. We observed that highest Jm occurs when glucose levels are low and that this is accompanied by noticeable fragmentation of mitochondrial network. Incubation at 0 mM and 3 mM glucose led to 91% and 71% increase in Jm rates, respectively. Incubation of INS-1E cells at 20 mM glucose did not cause any significant change to Jm generation rates and neither was observed any significant change in mitochondrial network structure. Altogether, our data suggest that GSIS has antioxidant character, and is accompanied with mitochondrial network reorganization. Supported by the grant No. 15-02022Y to A.D. from the grant Agency of the Czech Republic. Reference [1] Ježek J, Dlaskova A, Zelenka J, Jabůrek M, Ježek P. H2O2-Activated Mitochondrial Phospholipase iPLA2γ Prevents Lipotoxic Oxidative Stress in Synergy with UCP2, Amplifies Signaling via G-Protein-Coupled Receptor GPR40, and Regulates Insulin Secretion in Pancreatic β-Cells. Antioxid Redox Signal. 2015 23(12):958-72.