Physiological functions of the mitochondrial uncoupling proteins UCP2 and UCP3

Evidence for the physiological functions of UCP2 and UCP3 is critically reviewed. They do not mediate adaptive thermogenesis, but they may be significantly thermogenic under specific pharmacological conditions. There is strong evidence that the mild regulated uncoupling they cause attenuates mitochondrial ROS production, protects against cellular damage, and diminishes insulin secretion. Evidence that they export fatty acids physiologically is weak. UCP2 and UCP3 are important potential targets for treatment of aging, degenerative diseases, diabetes, and perhaps obesity

Calcium-dependent mitochondrial permeability transition is augmented in the kidney of Goto-Kakizaki diabetic rat

Renal disease associated with diabetes mellitus is a major problem among diabetic patients. The role of mitochondria in the pathogenesis of diabetes has received a large amount of attention in the last years, but many aspects of this subject are still poorly understood. In the present study, we studied the susceptibility of the mitochondrial permeability transition (MPT) on kidney mitochondria from the Goto-Kakizaki (GK) rat, an animal model featuring physiological and pathological alterations characteristic of type 2 diabetes.Kidney mitochondria were isolated by differential centrifugations; mitochondrial electric transmembrane potential and calcium loading capacity were evaluated with a TPP+-selective electrode and with a calcium-sensitive fluorescent probe. Coenzyme Q9, Q10 and vitamin E were evaluated by high-performance liquid chromatography (HPLC).Kidney mitochondria from the diabetic animals had an increased susceptibility to the induction of the MPT by calcium. We observed a loss of calcium-loading capacity and a higher calcium-induced mitochondrial depolarization. Vitamin E and coenzyme Q9 were also increased in kidney mitochondria from GK rats.The results show an enhanced MPT activation in kidney mitochondria from GK rats, which lead us to suggest that this condition may be one major alteration triggered by chronic diabetes in kidney cells, ultimately leading to cell dysfunction. Copyright © 2004 John Wiley & Sons, Ltd

Mitochondrial Physiology and Gene Expression Analyses Reveal Metabolic and Translational Dysregulation in Oocyte-Induced Somatic Nuclear Reprogramming

While reprogramming a foreign nucleus after somatic cell nuclear transfer (SCNT), the enucleated oocyte (ooplasm) must signal that biomass and cellular requirements changed compared to the nucleus donor cell. Using cells expressing nuclear-encoded but mitochondria-targeted EGFP, a strategy was developed to directly distinguish maternal and embryonic products, testing ooplasm demands on transcriptional and post-transcriptional activity during reprogramming. Specifically, we compared transcript and protein levels for EGFP and other products in pre-implantation SCNT embryos, side-by-side to fertilized controls (embryos produced from the same oocyte pool, by intracytoplasmic injection of sperm containing the EGFP transgene). We observed that while EGFP transcript abundance is not different, protein levels are significantly lower in SCNT compared to fertilized blastocysts. This was not observed for Gapdh and Actb, whose protein reflected mRNA. This transcript-protein relationship indicates that the somatic nucleus can keep up with ooplasm transcript demands, whilst transcription and translation mismatch occurs after SCNT for certain mRNAs. We further detected metabolic disturbances after SCNT, suggesting a place among forces regulating post-transcriptional changes during reprogramming. Our observations ascribe oocyte-induced reprogramming with previously unsuspected regulatory dimensions, in that presence of functional proteins may no longer be inferred from mRNA, but rather depend on post-transcriptional regulation possibly modulated through metabolism

Nuclear Reprogramming: Kinetics of Cell Cycle and Metabolic Progression as Determinants of Success

Establishment of totipotency after somatic cell nuclear transfer (NT) requires not only reprogramming of gene expression, but also conversion of the cell cycle from quiescence to the precisely timed sequence of embryonic cleavage. Inadequate adaptation of the somatic nucleus to the embryonic cell cycle regime may lay the foundation for NT embryo failure and their reported lower cell counts. We combined bright field and fluorescence imaging of histone H2b-GFP expressing mouse embryos, to record cell divisions up to the blastocyst stage. This allowed us to quantitatively analyze cleavage kinetics of cloned embryos and revealed an extended and inconstant duration of the second and third cell cycles compared to fertilized controls generated by intracytoplasmic sperm injection (ICSI). Compared to fertilized embryos, slow and fast cleaving NT embryos presented similar rates of errors in M phase, but were considerably less tolerant to mitotic errors and underwent cleavage arrest. Although NT embryos vary substantially in their speed of cell cycle progression, transcriptome analysis did not detect systematic differences between fast and slow NT embryos. Profiling of amino acid turnover during pre-implantation development revealed that NT embryos consume lower amounts of amino acids, in particular arginine, than fertilized embryos until morula stage. An increased arginine supplementation enhanced development to blastocyst and increased embryo cell numbers. We conclude that a cell cycle delay, which is independent of pluripotency marker reactivation, and metabolic restraints reduce cell counts of NT embryos and impede their development

Ubiquinone is not required for proton conductance by uncoupling protein 1 in yeast mitochondria.

Q (coenzyme Q or ubiquinone) is reported to be a cofactor obligatory for proton transport by UCPs (uncoupling proteins) in liposomes [Echtay, Winkler and Klingenberg (2000) Nature (London) 408, 609-613] and for increasing the binding of the activator retinoic acid to UCP1 [Tomás, Ledesma and Rial (2002) FEBS Lett. 526, 63-65]. In the present study, yeast ( Saccharomyces cerevisiae ) mutant strains lacking Q and expressing UCP1 were used to determine whether Q was required for UCP function in mitochondria. Wild-type yeast strain and two mutant strains (CENDeltaCOQ3 and CENDeltaCOQ2), both not capable of synthesizing Q, were transformed with the mouse UCP1 gene. UCP1 activity was measured as fatty acid-dependent, GDP-sensitive proton conductance in mitochondria isolated from the cells. The activity of UCP1 was similar in both Q-containing and -deficient yeast mitochondria. We conclude that Q is neither an obligatory cofactor nor an activator of proton transport by UCP1 when it is expressed in yeast mitochondria

Microfluidic Protocol for pre-implantation culture of single mammalian embryos: towards and optimal culture protocol

Microfluidics holds great potential for the field of assisted reproduction techniques (ART), to provide integrated platforms for combined embryo culture and characterization. The development of mouse embryos is not impaired in a microfluidic format: it proceeds faster during the pre-implantation period (< day 4), and gives similar birth rates. More importantly, single embryo culture is enhanced in a microfluidic environment

Microfluidic Protocol for pre-implantation culture of single mammalian embryos: towards and optimal culture protocol

Microfluidics holds great potential for the field of assisted reproduction techniques (ART), to provide integrated platforms for combined embryo culture and characterization. The development of mouse embryos is not impaired in a microfluidic format: it proceeds faster during the pre-implantation period (< day 4), and gives similar birth rates. More importantly, single embryo culture is enhanced in a microfluidic environment

Synergy of fatty acid and reactive alkenal activation of proton conductance through uncoupling protein 1 in mitochondria

The kinetics of proton transport through mammalian UCP1 (uncoupling protein 1) expressed in yeast mitochondria were measured. There was little or no UCP1 activity in the absence of added palmitate, but significant activity in its presence. The activator 4-HNE (4-hydroxy-2-nonenal) had little effect when added alone, but significantly enhanced proton conductance in the presence of added palmitate. Activation of the proton conductance of UCP1 was synergistic: proton conductance in the presence of both palmitate and 4-HNE was significantly greater than the sum of the individual effects. Mitochondria from control yeast transformed with empty vector showed no such synergy, showing that synergy is a property of UCP1. Activation by the 4-HNE analogue trans-cinnamate showed essentially the same characteristics as activation by 4-HNE. Mitochondria from brown adipose tissue also showed synergistic activation of GDP-sensitive proton conductance by palmitate and 4-HNE. These results show that reactive alkenals activate the proton conductance of UCP1 more strongly when fatty acids are also added, with implications for both mechanistic and physiological models of UCP1 activation

A signalling role for 4-hydroxy-2-nonenal in regulation of mitochondrial uncoupling

Oxidative stress and mitochondrial dysfunction are associated with disease and aging. Oxidative stress results from overproduction of reactive oxygen species (ROS), often leading to peroxidation of membrane phospholipids and production of reactive aldehydes, particularly 4-hydroxy-2-nonenal. Mild uncoupling of oxidative phosphorylation protects by decreasing mitochondrial ROS production. We find that hydroxynonenal and structurally related compounds (such as trans-retinoic acid, trans-retinal and other 2-alkenals) specifically induce uncoupling of mitochondria through the uncoupling proteins UCP1, UCP2 and UCP3 and the adenine nucleotide translocase (ANT). Hydroxynonenal-induced uncoupling was inhibited by potent inhibitors of ANT (carboxyatractylate and bongkrekate) and UCP (GDP). The GDP-sensitive proton conductance induced by hydroxynonenal correlated with tissue expression of UCPs, appeared in yeast mitochondria expressing UCP1 and was absent in skeletal muscle mitochondria from UCP3 knockout mice. The carboxyatractylate-sensitive hydroxynonenal stimulation correlated with ANT content in mitochondria from Drosophila melanogaster expressing different amounts of ANT. Our findings indicate that hydroxynonenal is not merely toxic, but may be a biological signal to induce uncoupling through UCPs and ANT and thus decrease mitochondrial ROS production

COMPARA\u106\uc3O FOTOINTERPRETATIVA ENTRE AEROFOTO e imagem de sat \ue9lite

This study evaluated the result of different methodologies in determining the use and occupancy of land using digital aerial photographs with spatial resolution of 1 m (scenario 01) and satellite imagery with a spatial resolution of 0.5 m ( scenario 02), photo-interpreted screen, on a scale of 1:2.000 mapping, watershed 'Horizonte' stream, in Alegre, ES state. We used the global performance indices and Kappa index for determining the average error between the sets and t test at 5 % probability to assess the degree of significance in the process of image interpretation between scenarios 01 and 02. We identified 27 classes of land use for scenarios 01 and 02. The classes of pasture (599.62 and 442.43 ha) and forest fragmentation (319.45 and 258.07 ha) respectively, occupying the largest areas in the two scenarios, composing 69.80 % for scenario 01 and 53.04 % for scenario 02 of the total area. There was a positive variation of 6.67 % in the DG scenario in scenarios 01 and 02. However, the K shows that the scenario 02 has a lower rate of errors of omission and commission of 2.09 %, but does not present significance by the statistical analysis presented at 5 % level by t test.Este estudo objetivou avaliar o uso de diferentes metodologias na determina\ue7\ue3o do uso e ocupa\ue7\ue3o da terra utilizando aerofotos digitais, com resolu\ue7\ue3o espacial de 1 m (cen\ue1rio 01) e imagens de sat\ue9lite, com resolu\ue7\ue3o espacial de 0,5 m (cen\ue1rio 02), fotointerpretadas em tela, na escala cartogr\ue1fica de 1:2.000, da bacia hidrogr\ue1fica do c\uf3rrego Horizonte, Alegre-ES. Utilizaram-se os \uedndices de desempenho global e \uedndice Kappa para determina\ue7\ue3o do erro m\ue9dio entre os cen\ue1rios e teste t a 5 % de probabilidade para avaliar o grau de signific\ue2ncia no processo de fotointerpreta\ue7\ue3o entre os cen\ue1rios 01 e 02. Foram identificadas 27 classes de uso da terra para os cen\ue1rios 01 e 02. As classes de pastagem (599,62 e 442,43 ha) e fragmento florestal (319,45 e 258,07 ha) respectivamente, ocupando maiores \ue1reas nos dois cen\ue1rios, compondo 69,80 % para o cen\ue1rio 01 e 53,04 % para o cen\ue1rio 02 das \ue1reas totais. Verificou-se uma varia\ue7\ue3o positiva do DG em 6,67 % do cen\ue1rio 01 em rela\ue7\ue3o ao cen\ue1rio 02. Entretanto o K demonstra que o cen\ue1rio 02 possui uma menor taxa entre os erros de omiss\ue3o e comiss\ue3o em 2,09 %, n\ue3o apresentado signific\ue2ncia pela an\ue1lise estat\uedstica em n\uedvel de 5 % pelo teste t