Papel del complejo IV en la expansión del tejido adiposo blanco asociada a la edad

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Medicina, Departamento de Bioquímica. Fecha de lectura: 21-01-2016Esta tesis tiene embargado el acceso al texto completo hasta el 21-07-2017El envejecimiento se ha asociado con la expansión del tejido adiposo blanco (WAT) así como con la disfunción mitocondrial, sin embargo los mecanismos moleculares implicados en ambos procesos se desconocen. Además, la mayoría de los estudios relacionados con la obesidad se han llevado a cabo empleando modelos de dieta rica en grasa (HFD) los cuales son mucho más exacerbados que la obesidad alcanzada durante el envejecimiento. En este trabajo demostramos que la expresión y actividad del complejo IV mitocondrial se reducen en los adipocitos blancos durante el envejecimiento, mientras que los niveles de expresión de otros marcadores mitocondriales no se alteran de manera significativa. En particular, mostramos que esta desestabilización del complejo IV durante el envejecimiento se inicia por un efecto primario del factor inducible por hipoxia 1 alfa (HIF1A) sobre la subunidad Vb del complejo IV (COX5B). Esta represión implica un sitio de unión canónico de c-MYC que se encuentra en el promotor de Cox5b, mientras que no está presente en el resto de subunidades del complejo IV. Así, hemos demostrado que c-MYC se une específicamente al promotor proximal de Cox5b y que esta unión disminuye notablemente en condiciones de hipoxia, asociadas a la estabilización de HIF1A. En cuanto a las consecuencias biológicas de la represión de COX5B, mostramos que el silenciamiento de COX5B in vitro promueve la desestabilización y reducción en la actividad del complejo IV, lo que promueve una disminución en la oxidación de ácidos grasos y una mayor acumulación de lípidos intracelulares. En este sentido, el silenciamiento de COX5B in vivo en WAT de ratones jóvenes aumenta el tamaño de los adipocitos, mientras que la restauración de la expresión COX5B en adipocitos de ratones envejecidos, contrarresta la expansión del WAT asociada a la edad. Por último, mostramos que la reducción en la expresión de COX5B dependiente de la edad también tiene lugar en humanos y que los niveles de expresión de esta subunidad pueden servir como marcador pronóstico para la respuesta a tratamientos de cirugía bariátrica en pacientes obesos. Nuestros hallazgos demuestran el papel central del complejo IV en general, y de COX5B en particular, durante la expansión de WAT asociada a la edad y que el restablecimiento de los niveles de expresión de COX5B en adipocitos envejecidos puede contrarrestar la expansión del WAT durante el envejecimiento.Aging is associated with white adipose tissue (WAT) enlargement and mitochondrial dysfunction but the molecular mechanisms involved are poorly understood. Moreover, most of the knowledge about obesity has been derived from studies using HFD conditions which are more severe than those associated to age-dependent WAT expansion. Here we show that mitochondrial complex IV expression and activity are reduced in white adipocytes during aging while other mitochondrial markers and mitochondrial content remain unchanged. This Cox5b repression is driven by oxygen sensing pathways and involves a canonical c-MYC binding site located at the Cox5b proximal promoter is not found in other complex IV subunits. Here we demonstrate that c-MYC binds specifically to the Cox5b proximal promoter and its binding is markedly repressed under hypoxia conditions, probably in a HIF1A dependent manner. COX5B knock down in vitro leads to a reduction in complex IV assembly and activity which favors a lower fatty acid oxidation and intracellular lipid accumulation. Local in vivo COX5B silencing in WAT of young mice increased adipocyte size, while restoration of COX5B expression in aging mice counteracted adipocyte size enlargement. Furthermore, age-dependent reduction of COX5B gene expression was also found in human WAT and COX5B expression levels could predict long-term weight loss after bariatric surgery treatment in obese patients. Our findings show a pivotal role of complex IV in white adipocyte enlargement and that restoration of COX5B expression prevents age-dependent WAT expansion

The Warburg Effect in Endothelial Cells and its Potential as an Anti-angiogenic Target in Cancer

Endothelial cells (ECs) make up the lining of our blood vessels and they ensure optimal nutrient and oxygen delivery to the parenchymal tissue. In response to oxygen and/or nutrient deprivation, ECs become activated and sprout into hypo-vascularized tissues forming new vascular networks in a process termed angiogenesis. New sprouts are led by migratory tip cells and extended through the proliferation of trailing stalk cells. Activated ECs rewire their metabolism to cope with the increased energetic and biosynthetic demands associated with migration and proliferation. Moreover, metabolic signaling pathways interact and integrate with angiogenic signaling events. These metabolic adaptations play essential roles in determining EC fate and function, and are perturbed during pathological angiogenesis, as occurs in cancer. The angiogenic switch, or the growth of new blood vessels into an expanding tumor, increases tumor growth and malignancy. Limiting tumor angiogenesis has therefore long been a goal for anticancer therapy but the traditional growth factor targeted anti-angiogenic treatments have met with limited success. In recent years however, it has become increasingly recognized that focusing on altered tumor EC metabolism provides an attractive alternative anti-angiogenic strategy. In this review, we will describe the EC metabolic signature and how changes in EC metabolism affect EC fate during physiological sprouting, as well as in the cancer setting. Then, we will discuss the potential of targeting EC metabolism as a promising approach to develop new anti-cancer therapies

Induction of the mitochondrial NDUFA4L2 protein by HIF-1α decreases oxygen consumption by inhibiting complex i activity

The fine regulation of mitochondrial function has proved to be an essential metabolic adaptation to fluctuations in oxygen availability. During hypoxia, cells activate an anaerobic switch that favors glycolysis and attenuates the mitochondrial activity. This switch involves the hypoxia-inducible transcription factor-1 (HIF-1). We have identified a HIF-1 target gene, the mitochondrial NDUFA4L2 (NADH dehydrogenase [ubiquinone] 1 alpha subcomplex, 4-like 2). Our results, obtained employing NDUFA4L2-silenced cells and NDUFA4L2 knockout murine embryonic fibroblasts, indicate that hypoxia-induced NDUFA4L2 attenuates mitochondrial oxygen consumption involving inhibition of Complex I activity, which limits the intracellular ROS production under low-oxygen conditions. Thus, reducing mitochondrial Complex I activity via NDUFA4L2 appears to be an essential element in the mitochondrial reprogramming induced by HIF-1This work was supported by Ministerio de Ciencia e Innovación (SAF 2007-06592, SAF2010-14851), Comunidad Autónoma de Madrid (SAL 2006/ 0311), Metoxia Project-Health (F2 2009-222741), and Recava Network (RD 06/0014/0031) to M.O.L.; PS09/00101 and CP07/00143 to A.M.-R.; PI060701, PS09/00116, and CP08/00204 to S.C.; BFU2008-03407/BMC to J.A.; SAF2009-08007 to J.A.E.; and CSD2007-00020 to A.M.-R. and J.A.E. The CNIC is supported by the Instituto de Salud Carlos III-MICINN and the Pro-CNIC Foundation. We are grateful to Mike Murphy (Mitochondrial Biology Unit, MRC, Cambridge, UK) for the gift of MitoQ. We also thank Stephen Y. Chan and Joseph Loscalzo (Harvard Medical School, Boston, MA) for providing us ISCU expression vector

MME+ fibro-adipogenic progenitors are the dominant adipogenic population during fatty infiltration in human skeletal muscle

Fatty infiltration, the ectopic deposition of adipose tissue within skeletal muscle, is mediated via the adipogenic differentiation of fibro-adipogenic progenitors (FAPs). We used single-nuclei and single-cell RNA sequencing to characterize FAP heterogeneity in patients with fatty infiltration. We identified an MME+ FAP subpopulation which, based on ex vivo characterization as well as transplantation experiments, exhibits high adipogenic potential. MME+ FAPs are characterized by low activity of WNT, known to control adipogenic commitment, and are refractory to the inhibitory role of WNT activators. Using preclinical models for muscle damage versus fatty infiltration, we show that many MME+ FAPs undergo apoptosis during muscle regeneration and differentiate into adipocytes under pathological conditions, leading to a reduction in their abundance. Finally, we utilized the varying fat infiltration levels in human hip muscles and found less MME+ FAPs in fatty infiltrated human muscle. Altogether, we have identified the dominant adipogenic FAP subpopulation in skeletal muscle.ISSN:2399-364

Acute Vhl Gene Inactivation Induces Cardiac HIF-Dependent Erythropoietin Gene Expression

Von Hippel Lindau (Vhl) gene inactivation results in embryonic lethality. The consequences of its inactivation in adult mice, and of the ensuing activation of the hypoxia-inducible factors (HIFs), have been explored mainly in a tissue-specific manner. This mid-gestation lethality can be also circumvented by using a floxed Vhl allele in combination with an ubiquous tamoxifen-inducible recombinase Cre-ER T2. Here, we characterize a widespread reduction in Vhl gene expression in Vhl floxed-UBC-Cre-ER T2 adult mice after dietary tamoxifen administration, a convenient route of administration that has yet to be fully characterized for global gene inactivation. Vhl gene inactivation rapidly resulted in a marked splenomegaly and skin erythema, accompanied by renal and hepatic induction of the erythropoietin (Epo) gene, indicative of the in vivo activation of the oxygen sensing HIF pathway. We show that acute Vhl gene inactivation also induced Epo gene expression in the heart, revealing cardiac tissue to be an extra-renal source of EPO. Indeed, primary cardiomyocytes and HL-1 cardiac cells both induce Epo gene expression when exposed to low O2 tension in a HIF-dependent manner. Thus, as well as demonstrating the potential of dietary tamoxifen administration for gene inactivation studies in UBC-Cre-ER T2 mouse lines

HIF1α-dependent uncoupling of glycolysis suppresses tumor cell proliferation

Hypoxia-inducible factor-1α (HIF1α) attenuates mitochondrial activity while promoting glycolysis. However, lower glycolysis is compromised in human clear cell renal cell carcinomas, in which HIF1α acts as a tumor suppressor by inhibiting cell-autonomous proliferation. Here, we find that, unexpectedly, HIF1α suppresses lower glycolysis after the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) step, leading to reduced lactate secretion in different tumor cell types when cells encounter a limited pyruvate supply such as that typically found in the tumor microenvironment in vivo. This is because HIF1α-dependent attenuation of mitochondrial oxygen consumption increases the NADH/NAD+ ratio that suppresses the activity of the NADH-sensitive GAPDH glycolytic enzyme. This is manifested when pyruvate supply is limited, since pyruvate acts as an electron acceptor that prevents the increment of the NADH/NAD+ ratio. Furthermore, this anti-glycolytic function provides a molecular basis to explain how HIF1α can suppress tumor cell proliferation by increasing the NADH/NAD+ ratio.ISSN:2666-3864ISSN:2211-124

PHD1 controls muscle mTORC1 in a hydroxylation-independent manner by stabilizing leucyl tRNA synthetase

Abstract mTORC1 is an important regulator of muscle mass but how it is modulated by oxygen and nutrients is not completely understood. We show that loss of the prolyl hydroxylase domain isoform 1 oxygen sensor in mice (PHD1KO) reduces muscle mass. PHD1KO muscles show impaired mTORC1 activation in response to leucine whereas mTORC1 activation by growth factors or eccentric contractions was preserved. The ability of PHD1 to promote mTORC1 activity is independent of its hydroxylation activity but is caused by decreased protein content of the leucyl tRNA synthetase (LRS) leucine sensor. Mechanistically, PHD1 interacts with and stabilizes LRS. This interaction is promoted during oxygen and amino acid depletion and protects LRS from degradation. Finally, elderly subjects have lower PHD1 levels and LRS activity in muscle from aged versus young human subjects. In conclusion, PHD1 ensures an optimal mTORC1 response to leucine after episodes of metabolic scarcity

<i>Erythropoietin</i> and <i>glucose transporter-1</i> gene expression in isolated primary cardiomyocytes in response to hypoxia.

<p>Isolated rat cardiomyocyte cultures were subjected to basal normoxic conditions and/or hypoxia (1% O₂) for 24 hours. <i>Epo</i> (A) and <i>Glut1</i> (B) expression was then analyzed by RT-PCR and normalized to that of <i>Hprt</i>. The data from four independent experiments are expressed as the change relative to the normoxic values. Statistical significance was assessed using a two-tailed paired t-test (*, p<0.05).</p

Gross appearance of tamoxifen-fed Vhl^floxed-Cre-ERT2 mice.

<p>(A) Vhl^wt-UBC-Cre-ER^T2 (n = 3), Vhl^floxed (n = 9) and Vhl^floxed-UBC-Cre-ER^T2 (n = 10) mice were administered tamoxifen as indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0022589#pone-0022589-g001" target="_blank">Figure 1</a> and the spleen/body weight ratio was then determined. Statistical significance was assessed using a two-tailed Student's t-test (*, p<0.05; **, p<0.01). Representative images of spleens (B), snouts (C) and paws (D) of Vhl^floxed-UBC-Cre-ER^T2 and control Vhl^floxed mice are shown.</p

<i>Erythropoietin</i> and <i>glucose transporter-1</i> gene expression in HL-1 cardiomyocyte cell line in response to activation of the oxygen-sensing HIF pathway.

<p>(A,B,C) HL-1 cells were transfected with a siRNA for <i>Hif1α</i> (siHIF1α) or a scrambled siRNA control (siSCR) and 24 hours after transfection, the cells were exposed to normoxic or hypoxic (1% O₂) conditions. The expression of <i>Epo</i>, <i>Glut1</i> and <i>Hif1α</i> was measured as described above and the data from three independent experiments are expressed as the change relative to the normoxic values. Statistical significance was assessed using a two-tailed Student's t-test (*, p<0.05).</p