Metabolic Syndrome and ß-Oxidation of Long-Chain Fatty Acids in the Brain, Heart, and Kidney Mitochondria.

We present evidence that metabolic syndrome (MetS) represents the postreproductive stage of the human postembryonic ontogenesis. Accordingly, the genes governing this stage experience relatively weak evolutionary selection pressure, thus representing the metabolic phenotype of distant ancestors with ß-oxidation of long-chain fatty acids (FAs) as the primary energy source. Mitochondria oxidize at high-rate FAs only when succinate, glutamate, or pyruvate are present. The heart and brain mitochondria work at a wide range of functional loads and possess an intrinsic inhibition of complex II to prevent oxidative stress at periods of low functional activity. Kidney mitochondria constantly work at a high rate and lack inhibition of complex II. We suggest that in people with MetS, oxidative stress is the central mechanism of the heart and brain pathologies. Oxidative stress is a secondary pathogenetic mechanism in the kidney, while the primary mechanisms are kidney hypoxia caused by persistent hyperglycemia and hypertension. Current evidence suggests that most of the nongenetic pathologies associated with MetS originate from the inconsistencies between the metabolic phenotype acquired after the transition to the postreproductive stage and excessive consumption of food rich in carbohydrates and a sedentary lifestyle.

Long-Chain and Medium-Chain Fatty Acids in Energy Metabolism of Murine Kidney Mitochondria.

Scientists have long established that fatty acids are the primary substrates for kidney mitochondria. However, to date we still do not know how long-chain and middle-chain fatty acids are oxidized at the mitochondrial level. Our previous research has shown that mitochondria from the heart, brain, and kidney oxidize palmitoylcarnitine at a high rate only in the presence of succinate, glutamate, or pyruvate. In this paper, we report properties of the isolated kidney mitochondria and how malate and succinate affect the oxidation of C16 and C8 acylcarnitines. The isolated kidney mitochondria contain very few endogenous substrates and require malate to oxidize pyruvate, glutamate, and C16 or C8 acylcarnitines. We discovered that with 10 µM of C16 or C8 acylcarnitines, low concentrations of malate (0.2 mM) or succinate (0.5 mM) enhance the States 4 and 3 respiratory rates several times. The highest respiration rates were observed with C16 or C8 acylcarnitines and 5 mM succinate mixtures. Results show that kidney mitochondria, unlike the heart and brain mitochondria, lack the intrinsic inhibition of succinate dehydrogenase. Additionally, results show that the oxidation of fatty acid by the small respirasome's supercomplex generates a high level of CoQH2, and this makes SDH in the presence of succinate reverse the flow of electrons from CoQH2 to reduce fumarate to succinate. Finally, we report evidence that succinate dehydrogenase is a key mitochondrial enzyme that allows fast oxidation of fatty acids and turns the TCA cycle function from the catabolic to the anabolic and anaplerotic metabolic pathways.

Physiological Levels of Nitric Oxide Diminish Mitochondrial Superoxide. Potential Role of Mitochondrial Dinitrosyl Iron Complexes and Nitrosothiols.

Mitochondria are the major source of superoxide radicals and superoxide overproduction contributes to cardiovascular diseases and metabolic disorders. Endothelial dysfunction and diminished nitric oxide levels are early steps in the development of these pathological conditions. It is known that physiological production of nitric oxide reduces oxidative stress and inflammation, however, the precise mechanism of "antioxidant" effect of nitric oxide is not clear. In this work we tested the hypothesis that physiological levels of nitric oxide diminish mitochondrial superoxide production without inhibition of mitochondrial respiration. In order to test this hypothesis we analyzed effect of low physiological fluxes of nitric oxide (20 nM/min) on superoxide and hydrogen peroxide production by ESR spin probes and Amplex Red in isolated rat brain mitochondria. Indeed, low levels of nitric oxide substantially attenuated both basal and antimycin A-stimulated production of reactive oxygen species in the presence of succinate or glutamate/malate as mitochondrial substrates. Furthermore, slow releasing NO donor DPTA-NONOate (100 µM) did not change oxygen consumption in State 4 and State 3. However, the NO-donor strongly inhibited oxygen consumption in the presence of uncoupling agent CCCP, which is likely associated with inhibition of the over-reduced complex IV in uncoupled mitochondria. We have examined accumulation of dinitrosyl iron complexes and nitrosothiols in mitochondria treated with fast-releasing NO donor MAHMA NONOate (10 µM) for 30 min until complete release of NO. Following treatment with NO donor, mitochondria were frozen for direct detection of dinitrosyl iron complexes using Electron Spin Resonance (ESR) while accumulation of nitrosothiols was measured by ferrous-N-Methyl-D-glucamine dithiocarbamate complex, Fe(MGD)2, in lysed mitochondria. Treatment of mitochondria with NO-donor gave rise to ESR signal of dinitrosyl iron complexes while ESR spectra of Fe(MGD)2 supplemented mitochondrial lysates showed presence of both dinitrosyl iron complexes and nitrosothiols. We suggest that nitric oxide attenuates production of mitochondrial superoxide by post-translational modifications by nitrosylation of protein cysteine residues and formation of protein dinitrosyl iron complexes with thiol-containing ligands and, therefore, nitric oxide reduction in pathological conditions associated with endothelial dysfunction may increase mitochondrial oxidative stress.

Identification of cancer stem cells and a strategy for their elimination.

It has been established previously that up to 40% of mouse CD34(+) hematopoietic stem cells are capable of internalizing exogenous dsDNA fragments both in vivo and ex vivo. Importantly, when mice are treated with a combination of cyclophosphamide and dsDNA, the repair of interstrand crosslinks in hematopoietic progenitors is attenuated, and their pluripotency is altered. Here we show for the first time that among various actively proliferating mammalian cell populations there are subpopulations capable of internalizing dsDNA fragments. In the context of cancer, such dsDNA-internalizing cell subpopulations display cancer stem cell-like phenotype. Furthermore, using Krebs-2 ascites cells as a model, we found that upon combined treatment with cyclophosphamide and dsDNA, engrafted material loses its tumor-initiating properties which we attribute to the elimination of tumor-initiating stem cell subpopulation or loss of its tumorigenic potential.

Mitochondrial oxidative phosphorylation in autosomal dominant optic atrophy.

BACKGROUND: Autosomal dominant optic atrophy (ADOA), a form of progressive bilateral blindness due to loss of retinal ganglion cells and optic nerve deterioration, arises predominantly from mutations in the nuclear gene for the mitochondrial GTPase, OPA1. OPA1 localizes to mitochondrial cristae in the inner membrane where electron transport chain complexes are enriched. While OPA1 has been characterized for its role in mitochondrial cristae structure and organelle fusion, possible effects of OPA1 on mitochondrial function have not been determined. RESULTS: Mitochondria from six ADOA patients bearing OPA1 mutations and ten ADOA patients with unidentified gene mutations were studied for respiratory capacity and electron transport complex function. Results suggest that the nuclear DNA mutations that give rise to ADOA in our patient population do not alter mitochondrial electron transport. CONCLUSION: We conclude that the pathophysiology of ADOA likely stems from the role of OPA1 in mitochondrial structure or fusion and not from OPA1 support of oxidative phosphorylation.

DNA polymerase eta contributes to strand bias of mutations of A versus T in immunoglobulin genes.

DNA polymerase (pol) eta participates in hypermutation of A:T bases in Ig genes because humans deficient for the polymerase have fewer substitutions of these bases. To determine whether polymerase eta is also responsible for the well-known preference for mutations of A vs T on the nontranscribed strand, we sequenced variable regions from three patients with xeroderma pigmentosum variant (XP-V) disease, who lack polymerase eta. The frequency of mutations in the intronic region downstream of rearranged J(H)4 gene segments was similar between XP-V and control clones; however, there were fewer mutations of A:T bases and correspondingly more substitutions of C:G bases in the XP-V clones (p < 10(-7)). There was significantly less of a bias for mutations of A compared with T nucleotides in the XP-V clones compared with control clones, whereas the frequencies for mutations of C and G were identical in both groups. An analysis of mutations in the WA sequence motif suggests that polymerase eta generates more mutations of A than T on the nontranscribed strand. This in vivo data from polymerase eta-deficient B cells correlates well with the in vitro specificity of the enzyme. Because polymerase eta inserts more mutations opposite template T than template A, it would generate more substitutions of A on the newly synthesized strand.

Expression of human AID in yeast induces mutations in context similar to the context of somatic hypermutation at G-C pairs in immunoglobulin genes.

BACKGROUND: Antibody genes are diversified by somatic hypermutation (SHM), gene conversion and class-switch recombination. All three processes are initiated by the activation-induced deaminase (AID). According to a DNA deamination model of SHM, AID converts cytosine to uracil in DNA sequences. The initial deamination of cytosine leads to mutation and recombination in pathways involving replication, DNA mismatch repair and possibly base excision repair. The DNA sequence context of mutation hotspots at G-C pairs during SHM is DGYW/WRCH (G-C is a hotspot position, R = A/G, Y = T/C, W = A/T, D = A/G/T). RESULTS: To investigate the mechanisms of AID-induced mutagenesis in a model system, we studied the genetic consequences of AID expression in yeast. We constructed a yeast vector with an artificially synthesized human AID gene insert using codons common to highly expressed yeast genes. We found that expression of the artificial hAIDSc gene was moderately mutagenic in a wild-type strain and highly mutagenic in an ung1 uracil-DNA glycosylase-deficient strain. A majority of mutations were at G-C pairs. In the ung1 strain, C-G to T-A transitions were found almost exclusively, while a mixture of transitions with 12% transversions was characteristic in the wild-type strain. In the ung1 strain mutations that could have originated from deamination of the transcribed stand were found more frequently. In the wild-type strain, the strand bias was reversed. DGYW/WRCH motifs were preferential sites of mutations. CONCLUSION: The results are consistent with the hypothesis that AID-mediated deamination of DNA is a major cause of mutations at G-C base pairs in immunoglobulin genes during SHM. The sequence contexts of mutations in yeast induced by AID and those of somatic mutations at G-C pairs in immunoglobulin genes are significantly similar. This indicates that the intrinsic substrate specificity of AID itself is a primary determinant of mutational hotspots at G-C base pairs during SHM.