Search

Diabetes is a silent disease. It is the 8th most common cause of death that does not hurt until it is too late and the disease has developed. Technology plays a vital role in managing diabetes and educating patients about importance of the treatment. The patient must be able to manage his blood glucose level. However, blood glucose level is measured sporadically as it causes important discomfort to the patient. Measuring glucose level in subcutaneous tissue is minimally invasive technique and thus considerably comfortable, but this level may be different from blood glucose level. We implemented a recently proposed method of blood glucose level calculation from the continuously measured subcutaneous tissue glucose level. Then, we developed a web portal that makes this method accessible to any doctor's office and any diabetic patient. To the best of our knowledge, we are the very first web portal that does this. In this paper, we describe the portal.

We aimed to study contribution of flavin IF, or ubiquinone IQ and IIIQ sites of superoxide formation by the respiratory chain (RC) complexes 1 and 3 in vivo in cancer (HepG2) and non-cancer cells (insulinoma INS-1E) using novel site-specific S1QEL and S3QEL suppressors1. Rates of superoxide release into the mitochondrial matrix (Jm) were semiquantified by MitoSOX Red fluorescence using confocal microscopy (for feasibility see2). Reflecting the IQ superoxide formation, S1QEL nearly completely suppressed rotenone-induced superoxide formation in glycolytic HepG2 cells but was inactive when these cells were forced to oxidative phosphorylation by aglycemia. Similarly, S1QEL was inefficient in INS-1E independently of the glucose dose. In contrast, with the decreasing glucose in INS-1E cells, the S3QEL suppressor indicated a lower superoxide fraction ascribed to the IIIQ site and higher remaining complex 1 IF site contribution, which correlated with higher NADH/NAD+. The export of redox equivalents by three mitochondrial shuttles was responsible for the observed NAD+ increase at higher glucose.

Calcium-independent phospholipases (iPLA2s) are a family of enzymes participating in cellular signaling by simultaneously producing free fatty acids and lysophospholipids. Phospholipase iPLA2γ (PNPLA8) is targeted to mitochondria and has been shown to augment GPR40-dependent insulin secretion in β-cell model insulinoma INS1-E cells [1]. Here, we investigated the participation of iPLA2γ on the blood glucose homeostasis and pancreatic β-cell insulin secretion using iPLA2γ-KO mice. Wild type (wt) controls and iPLA2γ-KO mice showed similar cholesterol, triglyceride and basal glucose levels after 6 hours of starving. However, the uric acid levels were increased in iPLA2γ-KOmice. Following the intraperitoneal injection of Intralipid, iPLA2γ-KO mice exhibited a prolonged hyperglycemic state compared to wt mice. Glucose-stimulated insulin release in isolated pancreatic islets (PI) was moderately decreased in iPLA2γ-KO PI. Physiologically relevant concentrations of palmitic acid stimulated insulin secretion in PI from wt mice, and this stimulation was absent in iPLA2γ-KO PI. In conclusion, the data are consistent with iPLA2γ ablation causing impairment of GPR40-dependent insulin secretion, resulting in prolonged glycemia. Thus, our results support the role of iPLA2γ in regulating insulin secretion in vivo.

Background Diabetic Goto Kakizaki (GK) rats represent an established model of type 2 diabetes that exhibit an onset of pancreatic islet (PI) pathology characterized by islet hypertrophy with a decreased number of insulin-secreting β-cells. Among the remaining β-cells, oxidative phosphorylation (OXPHOS) and consequently glucose-stimulated insulin secretion (GSIS) are impaired, perhaps owing to a deficit in mitochondrial DNA (mtDNA). We sought to identify this abnormality. Methods β-Cells were obtained from Accutase-dissolved PI isolated from GK or Wistar rats and sorted based on the positive Zn2+ signal of Newport Green. The mtDNA copy number per cell was quantified as the amplicon ratio by polymerase chain reaction using specific primers against the rat ND5 mt gene and UCP2 nuclear gene. Results The 12-month-old GK rats exhibited drastically reduced copy numbers per remaining β-cell, from 7,400 ± 600 in 12-month old Wistar rats (100%) to 24 ± 4%; mtDNA content in heart and liver was 70 ± 25% and 60 ± 20%, respectively. Versus age-paired Wistar rats, 6- and 4-month-old GK rats showed reductions to 60 ± 15% and 50 ± 20%, respectively. Conclusions OXPHOS of remnant β-cells in diabetic GK was drastically impaired due to the lack of sufficient mtDNA levels. We suggest the use of mtDNA quantification to quickly assess PI quality before transplantation.

Mitochondrial calcium-independent phospholipase A2γ (iPLA2γ) belongs to a family of enzymes that participate in cellular signaling by simultaneously producing free fatty acids and lysophospholipids. We have shown previously that the activity of iPLA2γ is increased following the addition of oxidants, such as tert-butyl hydroperoxide (tBHP) or H2O2. We have also shown that iPLA2γ-liberated fatty acids induce H+ transport mediated by mitochondrial uncoupling proteins and mitochondrial adenine nucleotide translocase (ANT) in heart, lung and spleen mitochondria, leading to a decrease in the mitochondrial protonmotive force and subsequent decrease in mitochondrial superoxide production [1,2]. Here we tested the hypothesis that iPLA2γ serves similar antioxidant function in brain. Using brain mitochondria isolated from wild-type (WT) mice, we found that low micromolar concentrations of tBHP induced increase in respiration and a parallel decrease in mitochondrial superoxide and H2O2 production. These effects of tBHP were fully prevented by bovine serum albumin and inhibited by R-bromoenol lactone, a selective inhibitor of iPLA2γ, and were absent in brain mitochondria isolated from iPLA2γ-KO mice. Moreover, the tBHP-induced effects were fully inhibited by carboxyatractyloside, indicating the participation of ANT. In addition, we observed decrease in mRNA levels of selected antioxidant enzymes and increase in lipid peroxidation products in brain tissues homogenates of iPLA2γ-KO mice compared to the WT controls. Our results support the hypothesis of an antioxidant role of iPLA2γ in brain, consistent with iPLA2γ-dependent, ANT-mediated attenuation of mitochondrial superoxide production. Supported by LTAUSA17174

Mitochondria of pancreatic β-cells act as the perfect glucose sensor substantiating glucose-stimulated insulin secretion (GSIS). Despite controversies, whether this leads or not to the increased superoxide release into the matrix, it has been recently indicated that H2O2 degradation is favored in the matrix upon GSIS [1]. We attempted to study this in model β-cells, INS-1E cells [2], using confocal microscopy time-resolved monitoring with Mito-HyPer. We found a substantial negative drift in fluorescence F485/F405 ratio, indicating degradation and possible slow H2O2 efflux in INS-1E cells pre-incubated with 3 mM or 11 mM glucose. After glucose addition to 25 mM, the H2O2 decrease was enhanced by several folds, which was prevented by inhibition of mitochondrial redox shuttles exporting NADPH to the cell cytosol on the expense of matrix NADH. We conclude that pancreatic β-cells do not create mitochondrial oxidative stress upon GSIS.

Oxidative stress of mitochondrial origin, i.e. elevated mitochondrial superoxide production, belongs to major factors determining aging and oxidative-stress-related diseases. Antioxidants, such as the mitochondria-targeted coenzyme Q, MitoQ(10), may prevent or cure these pathological conditions. To elucidate pro- and anti-oxidant action of MitoQ(10), we studied its effects on HepG2 cell respiration, mitochondrial network morphology, and rates of superoxide release (above that neutralized by superoxide dismutase) to the mitochondrial matrix (J(m)). MitoSOX Red fluorescence confocal microscopy monitoring of J(m) rates showed pro-oxidant effects of 3.5-fold increased J(m) with MitoQ(10). MitoQ(10) induced fission of the mitochondrial network which was recovered after 24h. In rotenone-inhibited HepG2 cells (i.e., already under oxidative stress) MitoQ(10) sharply decreased rotenone-induced J(m), but not together with the Complex II inhibitor thenoyltrifluoroacetone. Respiration of HepG2 cells and isolated rat liver mitochondria with MitoQ(10) increased independently of rotenone. The increase was prevented by thenoyltrifluoroacetone. These results suggest that MitoQ(10) accepts electrons prior to the rotenone-bound Q-site, and the Complex II reverse mode oxidizes MitoQ(10)H(2) to regenerate MitoQ(10). Consequently, MitoQ(10) has a pro-oxidant role in intact cells, whereas it serves as an antioxidant when Complex I-derived superoxide generation is already elevated due to electron flow retardation. Moreover, unlike mitochondrial uncoupling, MitoQ(10) exerted its antioxidant role when Complex I proton pumping was retarded by a hydrophobic amiloride, 5-(N-ethyl-N-isopropyl) amiloride. Consequently, MitoQ(10) may be useful in the treatment of diseases originating from impairment of respiratory chain Complex I due to oxidatively damaged mitochondrial DNA, when its targeted delivery to pathogenic tissues is ensured.

The recent knowledge on mitochondria as the substantial source of reactive oxygen species, namely superoxide and hydrogen peroxide efflux from mitochondria, is reviewed, as well as nitric oxide and subsequent peroxynitrite generation in mitochondria and their effects. The reactive oxygen species formation in extramitochondrial locations, in peroxisomes, by cytochrome P450, and NADPH oxidase reaction, is also briefly discussed. Conditions are pointed out under which mitochondria represent the major ROS source for the cell: higher percentage of non-phosphorylating and coupled mitochondria, in vivo oxygen levels leading to increased intensity of the reverse electron transport in the respiratory chain, and nitric oxide effects on the redox state of cytochromes. We formulate hypotheses on the crucial role of ROS generated in mitochondria for the whole cell and organism, in concert with extramitochondrial ROS and antioxidant defense. We hypothesize that a sudden decline of mitochondrial ROS production converts cells or their microenvironment into a "ROS sink" represented by the instantly released excessive capacity of ROS-detoxification mechanisms. A partial but immediate decline of mitochondrial ROS production may be triggered by activation of mitochondrial uncoupling, specifically by activation of recruited or constitutively present uncoupling proteins such as UCP2, which may counterbalance the mild oxidative stress.

Cancer metabolic alterations result from complex genetic and epigenetic adjustments which include mitochondrial pathways glutaminolysis, reductive carboxylation (RC), and 2-hydroxy-glutarate (2HG) production. We studied regulatory mechanisms that promote anabolic function of mitochondrial isocitrate dehydrogenase 2 (IDH2) towards RC and/or 2HG production in breast cancer cell lines, especially the activity of mitochondrial deacetylase sirtuin SIRT3, which was previously implicated in the regulation of IDH2 enzymatic activity. We demonstrated that IDH2 is a direct substrate of SIRT3 and that distinct regulation by SIRT3 of oxidative vs. reductive IDH2 activity exists. The analysis of metabolite labeling with stable isotope shows that mitochondrial production of 2HG by wild-type IDH2 is largely dependent on SIRT3 presence and activity, as assumed by overexpression of wild-type SIRT3 and SIRT3-inactive mutant in breast cancer cells. Moreover, we demonstrate that IDH2 activity produces 2HG in vitro, as assayed by the product analysis of the enzyme reaction of the isolated recombinant wild-type IDH2. The observed 2HG-producing activity is regulated by SIRT3, as an acetylation surrogate IDH2 mutant K413Q tend to decrease levels of 2HG in vitro. Our findings impact understanding of breast cancer etiology, since breast cancer cell lines exhibit a broad range of IDH2 expression and metabolic phenotypes, including 2HG levels.

Transport properties of plant mitochondria from potato tubers were investigated using the swelling technique and membrane potential measurements. Proton-dependent swelling of fatty acid-depleted mitochondria in potassium acetate with valinomycin was possible only in the presence of fatty acids (linoleic acid and 12-(4-azido-2-nitrophenylamino)dodecanoic acid) and was inhibited by various purine nucleotides including ATP, GDP, and GTP. Swelling representing uptake of hexanesulfonate was also inhibited by purine nucleotides. Also, the membrane potential of fatty acid-depleted potato mitochondria energized by succinate declined upon the addition of linoleic acid or 12-(4-azido-2-nitrophenylamino)dodecanoic acid, and this decrease was prevented by ATP and other purine nucleotides. These transport activities are identical to those reported for brown adipose tissue mitochondria and related to the uncoupling protein; therefore, we ascribed them to the plant mitochondrial uncoupling protein (PUMP). A major difference between plant and mammalian uncoupling protein is that PUMP transports small hydrophilic anions such as Cl- very slowly, if at all. We suggest that PUMP may play an important role in plant physiology, where a regulated uncoupling and thermogenesis can proceed during fruit and seed development.

Uncoupling protein mediates electrophoretic transport of protons and anions across the inner membrane of brown adipose tissue mitochondria. The mechanism and site of proton transport, the mechanism by which fatty acids activate proton transport, and the relationship between fatty acids and anion transport are unknown. We used fluorescent probes to measure H+ and anion transport in vesicles reconstituted with purified uncoupling protein and carried out a comparative study of the effects of laurate and its close analogue, undecanesulfonate. Undecanesulfonate was transported by uncoupling protein with a Km value similar to that observed for laurate as it activated H+ transport. Both laurate and undecanesulfonate inhibited Cl- with competitive kinetics. Undecanesulfonate inhibited laurate-induced H+ transport with competitive kinetics. Undecanesulfonate and laurate differed in two important respects. (i) Laurate caused uncoupling protein-mediated H+ transport, whereas undecanesulfonate did not. (ii) Lauric acid was rapidly transported across the bilayer by nonionic diffusion, whereas undecanesulfonic was not. We infer that the role of uncoupling protein in H+ transport is to transport fatty acid anions and that fatty acids induce H+ transport because they can diffuse electroneutrally across the membrane. According to this hypothesis, uncoupling protein is a pure anion porter and does not transport protons; rather it is designed to enable fatty acids to behave as cycling protonophores.

Calcium-independent phospholipase A2γ (iPLA2γ) catalyses the hydrolysis of fatty acids from phospholipids with the consequent generation of lysophospholipids. iPLA2γ is targeted to peroxisomes and mitochondria and results from our laboratory have shown that the activity of mitochondrial iPLA2γ is increased by a redox-sensitive process [1]. Here we aimed to characterize the properties of isolated recombinant iPLA2γ and test the hypothesis that iPLA2γ is activated by thiol oxidation directly. We expressed His-tagged human iPLA2γ using HepG2 or HEK293 cell lines and reconstituted recombinant purified iPLA2γ into liposomes composed of L-α-Phosphatitylcholine and cardiolipin. Using selected specific fluorescent probes and GC/MS analysis, we observed H2O2-induced release of fatty acids in liposomes containing purified recombinant iPLA2γ but not in liposomes lacking the enzyme. The H2O2-activated enzyme released free fatty acids with a specific activity of about 20 nmol.min-1.mg-1. The activity increased several fold in the presence of bovine serum albumin or in mixed micelles containing non-ionic detergent, indicating a possible inhibition of iPLA2γ by the products of its enzymatic activities. H2O2 activated iPLA2γ with a half-maximal constant corresponding to about 1.5 mol H2O2 per mol of enzyme and the active enzyme was fully inhibited by thiol reducing compounds. These results indicate that the regulation of iPLA2γ is dependent on reversible oxidation of accessible cysteine residues and also on a feedback regulation by either fatty acids or lysophospholipids, which is consistent with the physiological participation of iPLA2γ in cellular redox signaling.Supported by the grant GA15-02051S to M.J. from the Grant Agency of the Czech Republic.1. Jaburek M, Ježek J, Zelenka J, Ježek P. Antioxidant activity by a synergy of redox-sensitive mitochondrial phospholipase A2 and uncoupling protein-2 in lung and spleen. Int. J. Biochem & Cell Biol 45, 816-825, 2013