Energetic efficiency is important for health (e.g. genesis of obesity in humans), socio-economically important for meat production systems (e.g. feed cost to produce high quality protein) and important for the environment (e.g. use of natural resources and production of green house gases for meat production). Mitochondria are organelles that play an essential role in cellular metabolism and homeostasis related to energy utilization. These processes involve several proteins to ensure continuous availability of energy to the cells. The Shc proteins play a key role in substrate oxidation and energy metabolism. Additionally, the mitochondrial uncoupling proteins (UCPs) participate in physiological processes that may account for variation in energy expenditures in tissues. However, the mechanisms behind energy expenditure in animals are largely unknown. Thus, in order to study the energy metabolism and mitochondria function, studies using a nutritional, biochemical and molecular approaches were conducted with mice and cattle. The purpose of the first study was to determine if Shc proteins influence the metabolic response to acute (5-7 days) feeding of a high fat diet (HFD). To this end, whole animal energy expenditure and substrate oxidation were measured in the Shc knockout (ShcKO) and wild-type (WT) male mice consuming either a control or HFD diet. The activities of enzymes of glycolysis, the citric acid cycle, electron transport chain (ETC), and &beta;-oxidation were investigated in liver and skeletal muscle. The study showed that ShcKO increases (P < 0.05) energy expenditure (EE) adjusted for either total body weight or lean mass. This change in EE could explain the decrease in weight gain observed in ShcKO versus WT mice fed an HFD. Thus, our results indicate that Shc proteins should be considered as potential targets for developing interventions to mitigate weight gain on HFD by stimulating EE. Although decreased levels of Shc proteins influenced the activity of some enzymes in response to high fat feeding, such as increasing the activity of acyl-CoA dehydrogenase, it did not produce concerted changes in enzymes of glycolysis, citric acid cycle or the ETC. However, the physiological significance of these changes in enzyme activities remains to be determined. The purpose of experiment 2 was to study the association among heat production, blood parameters and mitochondrial DNA (mtDNA) copy number in Nellore bulls with high and low residual feed intake (RFI). The RFI values were obtained by regression of dry mater intake (DMI) in relation to average daily gain and mid-test metabolic body weight. Thus, 18 animals (9 in each group) were individually fed in a feedlot for 98 days. The heart rate (HR) of bulls was monitored for 4 consecutive days and used to calculate the estimated heat production (EHP). Electrodes were fitted to bulls with stretch belts and oxygen consumption was obtained using a facemask connected to the gas analyzer and HR was simultaneously measured for 15 minutes period. Daily EHP was calculated multiplying oxygen pulse (O2P) by the average HR, assuming 4.89 kcal/L of O2. Blood parameters such as hematocrit, hemoglobin, and glucose were assayed between 45 and 90 days. Immediately after slaughter, liver, muscle and adipose tissues (subcutaneous and visceral fat) were collected and, subsequently, mtDNA copy number per cell was quantified in tissues by quantitative real-time PCR. The proteome of hepatic tissue and levels of mitochondrial UCPs were also investigated. We found similar EHP and O2 consumption between RFI groups, while low RFI bulls (more efficient in feed conversion) shown lower HR, hemoglobin and hematocrit percentage (P < 0.05), confirming previous data from our group. In addition, 71 protein spots in liver were differentially expressed (P < 0.05) and no differences were detected for UCPs levels between RFI groups. Finally, there was no association between amounts of mtDNA and the RFI phenotypes, suggesting that mitochondrial abundance in liver, muscle, and adipose tissue was similar between efficient and inefficient groups. However, additional studies to confirm this hypothesis are needed. / A eficiência energética é importante para a saúde humana (gênese da obesidade), sistemas de produção de carne (custo dos alimentos para produzir proteínas de alta qualidade) e para o meio ambiente (uso de recursos naturais e mitigação de gases de efeito estufa). As mitocôndrias são organelas que desempenham papel central no metabolismo e homeostase relacionada a utilização da energia. Nas células, diversas proteínas são importantes para melhorar a eficiência energética. Como exemplos, as proteínas de sinalização Shc são fundamentais na oxidação de substratos e metabolismo energético e, nas mitocôndrias, existem as proteínas desacopladoras (UCPs), que participam do gasto energético e produção de calor. Entretanto, os mecanismos que controlam o gasto energético nos animais ainda é bastante desconhecido. Assim, para estudar o metabolismo energético e a função das mitocôndrias foram conduzidos dois estudos utilizando-se estratégias nutricionais, bioquímicas e moleculares com camundongos (1) e bovinos (2). Objetivou-se, no estudo 1, determinar se as proteínas Shc influenciam a resposta metabólica à alimentação contendo dieta rica em gordura (HFD) por 7 dias. Enzimas da via glicolítica, ciclo de Krebs, cadeia transportadora de elétron (CTE) e &beta;-oxidação foram analisadas no fígado e músculo de camundongos com baixa expressão de Shc (knockout ou ShcKO) e comuns (wild-type ou WT) submetidos à uma dieta controle ou à HFD. O gasto energético foi medido por câmara calorimétrica de respiração nos animais. O genótipo ShcKO apresentou maior gasto energético (P < 0.05) ajustado para o peso corporal total ou massa magra. Essa mudança poderia explicar o menor ganho de peso observado no genótipo ShcKO comparado ao WT quando consumindo a HFD. Esses resultados sugerem que as proteínas Shc podem contribuir no desenvolvimento de estratégias para mitigar o ganho de peso. Embora a redução dos níveis de Shc (ShcKO) tenha modificado a atividade de enzimas da &beta;-oxidação em resposta a HFD, tal condição não produziu mudanças semelhantes na via glicolítica, ciclo de Krebs ou CTE. Por isso, mais estudos são necessários para compreender a significância fisiológica dessas alterações. No experimento 2, objetivou-se estudar a associação entre produção de calor, variáveis sanguíneas e número de cópias de DNA mitocondrial (mtDNA) em bovinos Nelore agrupados pelo consumo alimentar residual (CAR). O CAR foi obtido por regressão do consumo de matéria seca em relação ao ganho de peso diário e peso metabólico do teste de desempenho (fase de crescimento). Assim, 18 bovinos (9 alto CAR versus 9 baixo CAR) foram confinados em baias individuais por 98 dias (fase de terminação). Os batimentos cardíacos (BC) dos bovinos foram monitorados por quatro dias consecutivos e, então, utilizados para o cálculo da produção de calor estimada (PCe). O consumo e pulso de oxigênio (O2) foram obtidos por meio de analisador de gás conectado à uma máscara facial, com medição simultânea dos BC por 15 minutos. A PCe diária foi calculada por multiplicação do pulso de O2 pela média dos BC, assumindo-se a constante 4.89 kcal/L de O2. Foram analisadas variáveis sanguíneas como hematócrito, hemoglobina e glicose (alto vs. baixo CAR). Imediatamente após o abate dos animais, amostras de fígado, músculo e tecido adiposo foram coletadas para determinação do mtDNA por PCR em tempo real. Adicionalmente, o proteoma do tecido hepático e os níveis de UCPs nos tecidos foram também investigados. Não houve diferença para PCe e consumo de O2 (P > 0.05) entre os grupos experimentais, entretanto, os animais baixo CAR (mais eficientes em conversão alimentar) demonstraram menor BC, concentração de hemoglobina e percentagem de hematócrito (P < 0.05), confirmando resultados previamente observados em nossos estudos. No fígado, 71 spots proteicos foram diferentes (P < 0.05) entre os grupos alto e baixo CAR, mas nenhuma diferença foi observada para os níveis de UCPs no músculo, fígado ou tecido adiposo. Por fim, não existiu diferença (P > 0.05) entre o número de cópias do mtDNA por célula entre os fenótipos estudados, sugerindo que o número de mitocôndrias e possivelmente a fosforilação oxidativa foi semelhante entre os grupos de animais eficientes e ineficientes. Contudo, são necessários estudos adicionais para confirmar essa hipótese.

La leucémie aiguë myéloïde (LAM) est une hémopathie maligne à progression rapide, qui se caractérise par une expansion clonale de précurseurs myéloïdes présentant un contrôle défectueux de leur prolifération et de leur différenciation. Une rémission complète peut être obtenue chez environ 80% à 85% des patients en associant cytarabine et anthracycline qui sont respectivement un inhibiteur de la synthèse d’ADN et un agent intercalant. Néanmoins, les résultats globaux pour les patients atteints de LAM restent médiocres et le taux de survie à 5 ans des patients âgés de plus de 60 ans, est inférieur à 10%. Le paradigme bien accepté de la leucémogenèse est que la leucémie résulte de la transformation d'une cellule unique appelée cellule souche leucémique (SCL) ou cellule initiatrice de leucémie (LIC) qui se développe par autorenouvellement et engendre par division asymétrique les blastes leucémiques bloqués dans leur différentiation. Les LIC vont être responsables du maintien et de la rechute de la maladie après le traitement chimiothérapeutique car si les traitements actuels sont relativement efficaces contre les blastes leucémiques, ils échouent au niveau des LIC. Un autre facteur important impliqué dans la résistance aux traitements est le microenvironnement de la moelle osseuse qui forme la niche hématopoïétique. Des études ont montré que différents composants cellulaires de la niche peuvent transférer des mitochondries à des cellules normales soumises à un stress métabolique ou dans un contexte pathologique, vers des cellules cancéreuses. Durant ma thèse, j'ai pu montrer que les cellules murines de la lignée MS-5 et/ou des cellules stromales primaires humaines dérivées de la moelle osseuse, utilisées comme cellules nourricières dans des expériences de co-culture, sont capables de transférer des mitochondries fonctionnelles aux cellules leucémiques. En utilisant différentes approches moléculaires et d'imagerie, nous avons pu montrer que les cellules de LAM peuvent, par ce transfert, augmenter leur masse mitochondriale jusqu'à 14%. Dans la co-culture, les cellules LAM receveuses ont montré une augmentation de 1,5 fois de la production d'adénosine triphosphate (ATP) mitochondriale et se sont révélées moins sujettes à une dépolarisation mitochondriale après chimiothérapie, affichant une survie plus élevée. Ce transfert unidirectionnel, renforcé par certains agents chimiothérapeutiques, nécessite des contacts cellule-cellule et semble se dérouler par une voie endocytaire qui reste à déterminer. Enfin, nous démontrons que le transfert mitochondrial est observé in vivo dans un modèle de xénogreffe de souris immunodéficientes NSG et se produit également dans les cellules et les progéniteurs initiateurs de la leucémie humaine et leur conférant une capacité plus élevée à initier des cultures leucémiques à long terme. Nous avons ainsi apporté la preuve qu'un transfert horizontal de mitochondries provenant des cellules stromales de la niche hématopoïétique participe aux phénomènes de chimiorésistance des cellules leucémiques receveuses. De ce fait, cibler ce transfert mitochondrial pourrait représenter une future cible thérapeutique originale pour un traitement adjuvant des LAM visant à interférer avec le soutien de leur microenvironnement. / Acute myelogenous leukemia (AML) is a heterogeneous group of hematopoietic malignancies arising from hematopoietic stem and/or progenitor cells that display defective control of their proliferation, differentiation, and maturation. Complete remission is achieved using anthracycline and cytarabine combination therapy in 80% to 85% of older patients. Nevertheless, the overall outcomes for AML patients remain poor, and the 5-year survival rate for patients over 60 is less than 10%. The well-accepted paradigm of leukemogenesis is that leukemia arises from the transformation of a single cell and is maintained by a small population of leukemic stem cells (LSC) or leukemia initiating cells (LICs). It is theorized that current treatments, although highly effective against the leukemic bulk, fail to eradicate the LICs that are therefore responsible for leukemia relapse. Another important factor involved in resistance to treatments is the microenvironment of the bone marrow, which is called the hematopoietic niche. Studies have shown that different niche cell components can transfer mitochondria to normal cells that undergo a metabolic stress and in a pathological context, to cancer cells. During my PhD we demonstrate that in an ex vivo niche-like coculture system, cells both primary and cultured AML cells take up functional mitochondria from murine or human bone marrow stromal cells. Using different molecular and imaging approaches, we show that AML cells can increase their mitochondrial mass by up to 14%. After coculture, recipient AML cells showed a 1.5-fold increase in mitochondrial ATP production and were less prone to mitochondrial depolarization after chemotherapy, displaying a higher survival. This unidirectional transfer enhanced by some chemotherapeutic agents required cell–cell contacts and proceed through an ill-defined endocytic pathway. Transfer was greater in AML blasts compared with normal cord blood CD34+ cells. Finally, we demonstrate that mitochondrial transfer was observed in vivo in an NSG immunodeficient mouse xenograft model and also occurred in human leukemia initiating cells and progenitors. As mitochondrial transfer provides a clear survival advantage following chemotherapy and a higher leukemic long-term culture initiating cell potential, targeting mitochondrial transfer could represent a future therapeutic target for AML treatment.

No description available.

Cell swelling may contribute to acute cell injury subsequent to ischemia/reperfusion. The potential role of mitochondrial uncoupling and the resultant mitochondrial swelling, due to opening of the mitochondrial permeability transition pore (MPTP), were examined in an in vitro ischemically pelleted isolated rabbit cardiomyocyte model using the protonophore, carbonyl cyanide m-chlorophenylhydrazone (CCCP) to uncouple mitochondria. Cyclosporin A (CsA) was employed to inhibit MPTP opening. Cell volume was determined by a cell-flotation, density-gradient assay, using bromododecane. Cell viability, subsequent to an osmotic stress, was determined by trypan blue permeability. Ischemic preconditioning (IPC) facilitated volume regulation following an osmotic stress. Ischemic-cell swelling was reduced by IPC. IPC protected ischemically pelleted cells, but CsA had no significant effects on injury or IPC protection. CCCP ischemia accelerated rates of ischemic contracture and injury, and abolished IPC protection. IPC protection was restored by CsA. In CCCP-ischemic-uncoupled cells, subjected to a reduced (170 mOsm) osmotic stress, CsA and IPC afforded independent and additive protection. Chelerythrine and 5-hydroxydecanoate (5-HD) blocked IPC, but did not reduce CsA protection. Electron microscopy confirmed that CCCP ischemia induced mitochondrial matrix swelling that was reduced by CsA. Cardioprotection by IPC and CsA was accompanied by proportional reductions in cell swelling. Morphometric analysis of the electron photomicrographs demonstrated that the mitochondrial volume fractions were significantly reduced in the CsA/CCCP (29.8 ± 2.3%, P < 0.004) and IPC/CsA/CCCP (31.5 ± 1.7%, P < 0.0008) groups as compared to the CCCP-ischemic group (40.5 ± 1.7%) The IPC/CCCP group (39.5 ± 4.2%) was not significantly different from the CCCP-ischemic group. NIM 811, a CsA analogue MPTP blocker with no calcineurin inhibitory activity, afforded protection similar to CsA. The results suggest that CsA protection may, in part, be mediated by reduction of mitochondrial swelling.

The cardiac toxicity of doxorubicin (DOX), a potent anticancer anthracycline antibiotic, is believed to be mediated through the generation of reactive oxygen species (ROS) in cardiomyocytes. This study aims to determine the function of cellular glutathione peroxidase (Gpx1), which is located in both mitochondria and cytosol, in defense against DOX-induced cardiomyopathy using a line of transgenic mice with cardiac overexpression of Gpx1. The Gpx1-overexpressing hearts were markedly more resistant than nontransgenic hearts to DOX-induced acute functional derangements, including impaired contractility and diastolic properties, decreased coronary flow rate, and reduced heart rate. In addition, DOX treatment impairs mitochondrial function of nontransgenic hearts as evident in a decreased rate of NAD-linked State 3 respiration, presumably a result of inactivation of complex I activity. This is associated with increases in the rates of NAD- and FAD-linked State 4 respiration and declines in P/O ratio, suggesting that the electron transfer and oxidative phosphorylation are uncoupled in these mitochondrial samples. These functional deficits of mitochondria could be largely prevented by Gpx1 overexpression. Taken together, these studies provide new evidence to further support the role of ROS, particularly H2O2 and/or fatty acid hydroperoxides, in causing contractile and mitochondrial dysfunction in mouse hearts acutely exposed to DOX.

Indiana University-Purdue University Indianapolis (IUPUI) / Because colorectal cancer (CRC) is the fourth most common cancer and the second leading cause of cancer death in the US, identifying biomarkers that might inform disease prevention and early diagnosis is of great public health importance. Mitochondria are key cytoplasmic organelles containing an independent genome, i.e., mitochondrial DNA (mtDNA). It has been increasingly recognized that mtDNA copy number (mtDNAcn) is a biomarker for mitochondrial function and cellular oxidative stress. To date, the few studies that have assessed associations between mtDNAcn and CRC outcomes have yielded inconsistent findings. Further, no epidemiologic study has examined the relationship between insulinemic potential of lifestyle and mtDNAcn. Therefore, in this dissertation, three studies were conducted using data from the Nurses’ Health Study and the Health Professionals Follow-Up Study. First, the association between pre-diagnostic leukocyte mtDNAcn and CRC risk was studied in a nested casecontrol study (324 cases/658 controls). Lower mtDNAcn was significantly associated with increased risk of CRC and proximal colon cancer. That inverse association remained significant among individuals with ≥ 8 years’ follow-up since blood collection, suggesting that mtDNAcn might serve as a long-term predictor of CRC risk. Second, possible associations of pre-diagnostic mtDNAcn with overall and CRC-specific survival were examined among 587 CRC patients. MtDNAcn was not significantly associated with survival overall or in subgroups by cancer location, grade, or stage. Among current smokers, there was an inverse association between one standard deviation (SD) decrease in mtDNAcn and increased overall death risk. Among patients diagnosed at or before 70.5 years of age and those with anti-inflammatory diets, reduced mtDNAcn was associated with lower CRC-specific death risk. Lastly, the cross-sectional association between empirical lifestyle index for hyperinsulinemia (ELIH) and mtDNAcn was investigated among 2,835 subjects without major chronic diseases (cancers, diabetes, and cardiovascular diseases). A significant inverse association was found: least-squares means ± SD of mtDNAcn z-score decreased dramatically across ELIH quintiles. Overall, the findings from this dissertation will contribute to the evaluation of mtDNAcn as a potential biomarker for CRC risk and prognosis, and inform future interventions designed to reduce the insulinemic potential of lifestyle factors to preserve mitochondrial function. / 2022-04-06

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第19175号 / 医博第4017号 / 新制||医||1010(附属図書館) / 32167 / 京都大学大学院医学研究科医学専攻 / (主査)教授 渡邊 直樹, 教授 松原 和夫, 教授 横出 正之 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Mitochondria are essential cellular organelles and the location of the electron transport chain, the site of the majority of energy production in the cell. Mitochondria contain their own circular genome approximately 16,000 base pairs in length. The mitochondrial genome (mtDNA) encodes 11 protein-coding genes essential for the electron transport chain, 22 tRNA genes, and two rRNA genes. Mitochondrial malfunction occurs in many diseases, and changes in the mitochondrial genome lead to numerous disorders. Multiple mitochondrial haplotypes and sequence features are associated with Alzheimer's disease. In this dissertation we utilized TreeScanning, an evolutionary-based haplotype approach to identify haplotypes and sequence variation associated with specific phenotypes: Alzheimer's disease case-control status, mitochondrial copy number, and 16 neuroimaging phenotypes related to Alzheimer's disease neurodegeneration. In the first two studies we utilized 1007 complete mitochondrial genomes from participants in the Cache County Study on Memory Health and Aging. First, individuals with mitochondrial haplotypes H6A1A and H6A1B showed a reduced risk of AD. Our study is the largest to date and the only study with complete mtDNA genome sequence data. Next, each cell contains multiple mitochondria, and each mitochondrion contains multiple copies of its own circular genome. The ratio of mitochondrial genomes to nuclear genomes is referred to as mitochondrial copy number. Decreases in mitochondrial copy number are known to occur in many tissues as people age, and in certain diseases. Three variants belonging to mitochondrial haplogroups U5A1 and T2 were significantly associated with higher mitochondrial copy number in our dataset. Each of these three variants was associated with higher mitochondrial copy number and we suggest several hypotheses for how these variants influence mitochondrial copy number by interacting with known regulators of mitochondrial copy number. Our results are the first to report sequence variation in the mitochondrial genome that lead to changes in mitochondrial copy number. The identification of these variants that increase mtDNA copy number has important implications in understanding the pathological processes that underlie these phenotypes. Lastly, we used an endophenotype-based approach to further characterize mitochondrial genetic variation and its relationship to risk markers for Alzheimer's disease. We analyzed longitudinal data from non-demented, mild cognitive impairment, and late onset Alzheimer's disease participants in the Alzheimer's Disease Neuroimaging Initiative with genetic, brain imaging, and behavioral data. Four clades were associated with three different endophenotypes: whole brain volume, percent change in temporal pole thickness, and left hippocampal atrophy over two years. This was the first study of its kind to identify mitochondrial variation associated with brain imaging endophenotypes of Alzheimer's disease. Together, these projects provide evidence of mtDNA involvement in the risk and physiological changes of Alzheimer's disease.

No description available.

Obesity is associated with reduced skeletal muscle insulin sensitivity, a major risk factor for development of type II diabetes. These metabolic diseases are commonly associated with an accumulation of mitochondrial dysfunction, which is speculated to contribute toward insulin resistance. High-fat diets reduce human skeletal muscle insulin sensitivity and mitochondrial function. Conversely, endurance training increases insulin sensitivity and enhances mitochondrial performance. Recent evidence in mice has found that central mechanisms of mitochondrial quality control, autophagy and mitophagy, may be suppressed in response to excess fat intake, but upregulated following endurance exercise training. These data may provide a mechanism for dietary and exercise-mediated regulation of mitochondrial quality and metabolic function. The current study investigated the impact of an acute high-fat diet on skeletal muscle autophagy and mitophagy in sedentary, healthy, non-obese college age males'. The expression of skeletal muscle autophagy and mitophagy protein markers were analyzed in response to a high-fat meal before and after a 5-day high-fat diet. Next, we examined the differences in skeletal muscle autophagy and mitophagy protein markers, and associations with skeletal muscle metabolic flexibility between endurance-trained male runners' and sedentary, healthy, non-obese males' following an overnight fast and in response to a high-fat meal. Autophagy markers' indicated reduced autophagy activity in response to a high-fat meal and following a high-fat diet, which exacerbated the high-fat meal response. However, these data could not be confirmed due to methodological limitations. Mitophagy markers were not significantly affected by the high-fat meal or diet. There were no significant differences in the expression of autophagy protein markers between endurance-trained and sedentary groups', but mitophagy markers were significantly elevated in endurance-trained runners'. Metabolic flexibility was not significantly different between groups' following an overnight fast or in response to a high-fat meal, and was not associated with the expression of autophagy and mitophagy protein markers. In conclusion, autophagy may be suppressed by a 5-day high-fat diet, but further analysis is required for confirmation. Endurance-trained male runners show increased markers of mitophagy, which were not associated with improved metabolic flexibility while fasted or following a high-fat meal. / Ph. D.