Jaroslav Zelenka

Antioxidant activity by a synergy of redox-sensitive mitochondrial phospholipase A2 and uncoupling protein-2 in lung and spleen.

Mitochondrial uncoupling protein-2 (UCP2) has been suggested to participate in the attenuation of the reactive oxygen species production, but the mechanism of action and the physiological significance of UCP2 activity remain controversial. Here we tested the hypothesis that UCP2 provides feedback downregulation of oxidative stress in vivo via synergy with an H2O2-activated mitochondrial calcium-independent phospholipase A2 (mt-iPLA2). Tert-butylhydroperoxide or H2O2 induced free fatty acid release from mitochondrial membranes as detected by gas chromatography/mass spectrometry, which was inhibited by r-bromoenol lactone (r-BEL) but not by its stereoisomer s-BEL, suggesting participation of mt-iPLA2γ isoform. Tert-butylhydroperoxide or H2O2 also induced increase in respiration and decrease in mitochondrial membrane potential in lung and spleen mitochondria from control but not UCP2-knockout mice. These data suggest that mt-iPLA2γ-dependent release of free fatty acids promotes UCP2-dependent uncoupling. Upon such uncoupling, mitochondrial superoxide formation decreased instantly also in the s-BEL presence, but not when mt-iPLA2 was blocked by R-BEL and not in mitochondria from UCP2-knockout mice. Mt-iPLA2γ was alternatively activated by H2O2 produced probably in conjunction with the electron-transferring flavoprotein:ubiquinone oxidoreductase (ETFQOR), acting in fatty acid β-oxidation. Palmitoyl-d,l-carnitine addition to mouse lung mitochondria, respiring with succinate plus rotenone, caused a respiration increase that was sensitive to r-BEL and insensitive to s-BEL. We thus demonstrate for the first time that UCP2, functional due to fatty acids released by redox-activated mt-iPLA2γ, suppresses mitochondrial superoxide production by its uncoupling action. In conclusion, H2O2-activated mt-iPLA2γ and UCP2 act in concert to protect against oxidative stress.

Reductive carboxylation and 2-hydroxyglutarate formation by wild-type IDH2 in breast carcinoma cells

Mitochondrial NADPH-dependent isocitrate dehydrogenase, IDH2, and cytosolic IDH1, catalyze reductive carboxylation of 2-oxoglutarate. Both idh2 and idh1 monoallelic mutations are harbored in grade 2/3 gliomas, secondary glioblastomas and acute myeloid leukemia. Mutant IDH1/IDH2 enzymes were reported to form an oncometabolite r-2-hydroxyglutarate (2HG), further strengthening malignancy. We quantified CO2-dependent reductive carboxylation glutaminolysis (RCG) and CO2-independent 2HG production in HTB-126 and MDA-MB-231 breast carcinoma cells by measuring (13)C incorporation from 1-(13)C-glutamine into citrate, malate, and 2HG. For HTB-126 cells, (13)C-citrate, (13)C-malate, and (13)C-2-hydroxyglutarate were enriched by 2-, 5-, and 15-fold at 5mM glucose (2-, 2.5-, and 13-fold at 25 mM glucose), respectively, after 6 h. Such enrichment decreased by 6% with IDH1 silencing, but by 30-50% upon IDH2 silencing while cell respiration and ATP levels rose up to 150%. Unlike 2HG production RCG declined at decreasing CO2. At hypoxia (5% O2), IDH2-related and unrelated (13)C-accumulation into citrate and malate increased 1.5-2.5-fold with unchanged IDH2 expression; whereas hypoxic 2HG formation did not. (13)C-2HG originated by ∼50% from other than IDH2 or IDH1 reactions, substantiating remaining activity in IDH1&2-silenced cells. Relatively high basal (12)C-2HG levels existed (5-fold higher vs. non-tumor HTB-125 cells) and (13)C-2HG was formed despite the absence of any idh2 and idh1 mutations in HTB-126 cells. Since RCG is enhanced at hypoxia (frequent in solid tumors) and 2HG can be formed without idh1/2 mutations, we suggest 2HG as an analytic marker (in serum, urine, or biopsies) predicting malignancy of breast cancer in all patients.

Delta Cell Hyperplasia in Adult Goto-Kakizaki (GK/MolTac) Diabetic Rats

Reduced beta cell mass in pancreatic islets (PI) of Goto-Kakizaki (GK) rats is frequently observed in this diabetic model, but knowledge on delta cells is scarce. Aiming to compare delta cell physiology/pathology of GK to Wistar rats, we found that delta cell number increased over time as did somatostatin mRNA and delta cells distribution in PI is different in GK rats. Subtle changes in 6-week-old GK rats were found. With maturation and aging of GK rats, disturbed cytoarchitecture occurred with irregular beta cells accompanied by delta cell hyperplasia and loss of pancreatic polypeptide (PPY) positivity. Unlike the constant glucose-stimulation index for insulin PI release in Wistar rats, this index declined with GK age, whereas for somatostatin it increased with age. A decrease of GK rat PPY serum levels was found. GK rat body weight decreased with increasing hyperglycemia. Somatostatin analog octreotide completely blocked insulin secretion, impaired proliferation at low autocrine insulin, and decreased PPY secretion and mitochondrial DNA in INS-1E cells. In conclusion, in GK rats PI, significant local delta cell hyperplasia and suspected paracrine effect of somatostatin diminish beta cell viability and contribute to the deterioration of beta cell mass. Altered PPY-secreting cells distribution amends another component of GK PIs pathophysiology.

Import of desired nucleic acid sequences using addressing motif of mitochondrial ribosomal 5S-rRNA for fluorescent in vivo hybridization of mitochondrial DNA and RNA

Based on the matrix-addressing sequence of mitochondrial ribosomal 5S-rRNA (termed MAM), which is naturally imported into mitochondria, we have constructed an import system for in vivo targeting of mitochondrial DNA (mtDNA) or mt-mRNA, in order to provide fluorescence hybridization of the desired sequences. Thus DNA oligonucleotides were constructed, containing the 5′-flanked T7 RNA polymerase promoter. After in vitro transcription and fluorescent labeling with Alexa Fluor® 488 or 647 dye, we obtained the fluorescent “L-ND5 probe” containing MAM and exemplar cargo, i.e., annealing sequence to a short portion of ND5 mRNA and to the light-strand mtDNA complementary to the heavy strand nd5 mt gene (5′-end 21 base pair sequence). For mitochondrial in vivo fluorescent hybridization, HepG2 cells were treated with dequalinium micelles, containing the fluorescent probes, bringing the probes proximally to the mitochondrial outer membrane and to the natural import system. A verification of import into the mitochondrial matrix of cultured HepG2 cells was provided by confocal microscopy colocalizations. Transfections using lipofectamine or probes without 5S-rRNA addressing MAM sequence or with MAM only were ineffective. Alternatively, the same DNA oligonucleotides with 5′-CACC overhang (substituting T7 promoter) were transcribed from the tetracycline-inducible pENTRH1/TO vector in human embryonic kidney T-REx®-293 cells, while mitochondrial matrix localization after import of the resulting unlabeled RNA was detected by PCR. The MAM-containing probe was then enriched by three-order of magnitude over the natural ND5 mRNA in the mitochondrial matrix. In conclusion, we present a proof-of-principle for mitochondrial in vivo hybridization and mitochondrial nucleic acid import.

Import of Fluorescent RNA into Mitochondria of Living Cells.

Methods of in vivo visualization and manipulation of mitochondrial genetic machinery are limited due to the need to surpass not only the cytoplasmic membrane but also two mitochondrial membranes. Here, we employ the matrix-addressing sequence of mitochondrial ribosomal 5S-rRNA (termed MAM), which is naturally imported into mammalian mitochondria, to construct an import system for in vivo targeting of mitochondrial (mt) DNA or mtRNA, in order to provide fluorescence hybridization of the desired sequences.