Antagonism between wild-type and mutant ß-catenin controls hepatoblastoma differentiation via fascin-1.

Background & Aims: ß-catenin is a well-known effector of the Wnt pathway, and a key player in cadherin-mediated cell adhesion. Oncogenic mutations of ß-catenin are very frequent in paediatric liver primary tumours. Those mutations are mostly heterozygous, which allows the co-expression of wild-type (WT) and mutated ß-catenins in tumour cells. We investigated the interplay between WT and mutated ß-catenins in liver tumour cells, and searched for new actors of the ß-catenin pathway. Methods: Using an RNAi strategy in ß-catenin-mutated hepatoblastoma (HB) cells, we dissociated the structural and transcriptional activities of ß-catenin, which are carried mainly by WT and mutated proteins, respectively. Their impact was characterised using transcriptomic and functional analyses. We studied mice that develop liver tumours upon activation of ß-catenin in hepatocytes (APCKO and ß-cateninΔexon3 mice). We used transcriptomic data from mouse and human HB specimens, and used immunohistochemistry to analyse samples. Results: We highlighted an antagonistic role of WT and mutated ß-catenins with regard to hepatocyte differentiation, as attested by alterations in the expression of hepatocyte markers and the formation of bile canaliculi. We characterised fascin-1 as a transcriptional target of mutated ß-catenin involved in tumour cell differentiation. Using mouse models, we found that fascin-1 is highly expressed in undifferentiated tumours. Finally, we found that fascin-1 is a specific marker of primitive cells including embryonal and blastemal cells in human HBs. Conclusions: Fascin-1 expression is linked to a loss of differentiation and polarity of hepatocytes. We present fascin-1 as a previously unrecognised factor in the modulation of hepatocyte differentiation associated with ß-catenin pathway alteration in the liver, and as a new potential target in HB. Impact and implications: The FSCN1 gene, encoding fascin-1, was reported to be a metastasis-related gene in various cancers. Herein, we uncover its expression in poor-prognosis hepatoblastomas, a paediatric liver cancer. We show that fascin-1 expression is driven by the mutated beta-catenin in liver tumour cells. We provide new insights on the impact of fascin-1 expression on tumour cell differentiation. We highlight fascin-1 as a marker of immature cells in mouse and human hepatoblastomas.

Silencing of RND3/RHOE inhibits the growth of human hepatocellular carcinoma and is associated with reversible senescence.

Rnd3/RhoE is an atypical Rho GTPase family member, known to be deregulated in many types of cancer. Previously, we showed that RND3 expression is downregulated in hepatocellular carcinoma (HCC) cell lines and tissues. In cancer cells, Rnd3 is involved in the regulation of cell proliferation and cell invasion. The implication of Rnd3 in HCC invasion was importantly studied whereas its role in cell growth needs further investigation. Thus, in this work, we aimed to better understand the impact of Rnd3 on tumor hepatocyte proliferation. Our results indicate that the silencing of RND3 induces a cell growth arrest both in vitro in 2D and 3D culture conditions and in vivo in tumor xenografts. The growth alteration after RND3 silencing in HCC cells is not due to an increase of cell death but to the induction of senescence. This RND3 knockdown-mediated phenomenon is dependent on the decrease of hTERT expression. Interestingly, after re-expression of RND3, these cells are able to bypass senescence and regain the ability to proliferate, with a re-expression of hTERT. Given that a low expression of Rnd3 is linked to the presence of satellite nodules in HCC, the transient senescence state observed might play a role in cancer progression.

La ética en el encuentro. Reflexiones a partir de la instrumentación del Consentimiento Informado en investigaciones cualitativas / Ethics in the encounter with others. Reflections on the implementation of Informed Consent (IC) in qualitative research / A ética no encontro. Reflexões a partir da instrumentação do Termo de Consentimento Livre e Esclarecido em pesquisa qualitativa

Resumen: En este artículo se reflexiona sobre la instrumentación del Consentimiento Informado (CI) en investigaciones cualitativas a partir de experiencias de un colectivo de investigadoras de la Facultad de Psicología de la Universidad de la República (Uruguay). Se abordan aspectos históricos sobre el origen del CI contextualizado en los Comités de Ética, ubicando la situación de la Facultad de Psicología de la Universidad de la República. Luego, se profundiza en la ética y en el CI como procesos reflexivos y dialógicos, más allá de los aspectos formales establecidos institucionalmente, y sus particularidades en investigaciones cualitativas desde la Psicología Social Comunitaria. A partir de situaciones particulares, ligadas a investigaciones realizadas por integrantes del colectivo, se reflexiona sobre: a) el rol de las instituciones en la implementación del CI en personas con historias de institucionalización. b) el anonimato y la posibilidad de coautoría. La introducción del CI no es inocua, por ello se piensa en los sentidos que estos procedimientos portan, su incidencia en los vínculos que se establecen en el proceso investigativo y su relación con la ética en la investigación. Las conclusiones versan sobre la necesidad de promover institucionalmente mecanismos para la reflexión, evitando la burocratización de la ética en los procesos de investigación, así como la importancia de la flexibilización y creatividad en cuanto a formas de dar consentimiento en contextos comunitarios.

Abstract: This paper presents a reflection about the implementation of Informed Consent (IC) in qualitative research, based on the experiences of a women researchers group from Facultad de Psicología de la Universidad de la República (Uruguay). In a first instance, a brief historical description is performed, regarding the origins of Ethics Committees and IC in general and in Facultad de Psicología de la UdelaR in particular. Then, an ethics and Informed Consent instrumentation analysis is elaborated, as a reflexive and dialogic process beyond the formal aspects institutionally established, and it's particularities in qualitative research from a Community Social Psychology approach. Through the narration of particular situations related to research project developed by members of the group, we reflect on: a) the role of institutions during the implementation of IC in people with institutionalization process. b) anonymity and co-authorship possibilities. IC's implementation is not an innocuous process, thus the necessity of exploring the meanings that these procedures carry, their incidence on the relationships that are established within research process and its link with research ethics. In the concluding section, the need of institutional devices to promote mechanisms for reflexive vigilance is presented, in order to avoid the bureaucratization of ethics in research, as well as the importance of flexibility and creativity in community consent contexts.

Resumo: Neste artigo, refletimos sobre a implementação do Consentimento Livre e Esclarecido (TC) em pesquisa qualitativa com base nas experiências de um grupo de pesquisadores da Faculdade de Psicologia da Universidade da República (Uruguai). Aspectos históricos sobre a origem do CI contextualizado nos Comitês de Ética são abordados, situando a situação da Faculdade de Psicologia da Universidade da República. Em seguida, a ética e o CI são aprofundados como processos reflexivos e dialógicos, além dos aspectos formais estabelecidos institucionalmente, e suas peculiaridades na pesquisa qualitativa da Psicologia Social Comunitária. A partir de situações particulares, vinculadas a pesquisas realizadas por membros do coletivo, refletimos sobre: a) o papel das instituições na implementação do CI em pessoas com histórias de institucionalização. b) anonimato e possibilidade de coautoria. A introdução do QI não é inócua, pois pensamos sobre os significados que esses procedimentos carregam, sua incidência nos vínculos que se estabelecem no processo investigativo e sua relação com a ética na investigação. As conclusões são sobre a necessidade de promover institucionalmente mecanismos de reflexão, evitando a burocratização da ética nos processos de pesquisa, bem como a importância da flexibilidade e criatividade em termos de formas de dar consentimento em contextos comunitários.

Endogenous IL-33 Contributes to Kidney Ischemia-Reperfusion Injury as an Alarmin.

Inflammation is a prominent feature of ischemia-reperfusion injury (IRI), which is characterized by leukocyte infiltration and renal tubular injury. However, signals that initiate these events remain poorly understood. We examined the role of the nuclear alarmin IL-33 in tissue injury and innate immune response triggered by experimental kidney ischemia-reperfusion. In wild-type mice, we found that IL-33 was constitutively expressed throughout the kidney in peritubular and periglomerular spaces, mainly by microvascular endothelial cells, from which it was released immediately during IRI. Compared with wild-type mice, mice lacking IL-33 (IL-33Gt/Gt) exhibited reductions in early tubular cell injury and subsequent renal infiltration of IFN-γ/IL-17A-producing neutrophils, with preservation of renal functions. This protection associated with decreased renal recruitment of myeloid dendritic cells, natural killer (NK) cells, and invariant natural killer T (iNKT) cells, the latter of which were reported as deleterious in IRI. Increases in the level of circulating IL-12, a key IL-33 cofactor, and the expression of ST2, an IL-33-specific receptor, on the surface of iNKT cells preceded the IL-33- and iNKT cell-dependent phase of neutrophil infiltration. Furthermore, IL-33 directly targeted iNKT cells in vitro, inducing IFN-γ and IL-17A production. We propose that endogenous IL-33 is released as an alarmin and contributes to kidney IRI by promoting iNKT cell recruitment and cytokine production, resulting in neutrophil infiltration and activation at the injury site. Our findings show a novel molecular mediator contributing to innate immune cell recruitment induced by renal ischemia-reperfusion and may provide therapeutic insights into AKI associated with renal transplantation.

Insulin receptor substrate signaling suppresses neonatal autophagy in the heart.

The induction of autophagy in the mammalian heart during the perinatal period is an essential adaptation required to survive early neonatal starvation; however, the mechanisms that mediate autophagy suppression once feeding is established are not known. Insulin signaling in the heart is transduced via insulin and IGF-1 receptors (IGF-1Rs). We disrupted insulin and IGF-1R signaling by generating mice with combined cardiomyocyte-specific deletion of Irs1 and Irs2. Here we show that loss of IRS signaling prevented the physiological suppression of autophagy that normally parallels the postnatal increase in circulating insulin. This resulted in unrestrained autophagy in cardiomyocytes, which contributed to myocyte loss, heart failure, and premature death. This process was ameliorated either by activation of mTOR with aa supplementation or by genetic suppression of autophagic activation. Loss of IRS1 and IRS2 signaling also increased apoptosis and precipitated mitochondrial dysfunction, which were not reduced when autophagic flux was normalized. Together, these data indicate that in addition to prosurvival signaling, insulin action in early life mediates the physiological postnatal suppression of autophagy, thereby linking nutrient sensing to postnatal cardiac development.

UCP3 regulates cardiac efficiency and mitochondrial coupling in high fat-fed mice but not in leptin-deficient mice.

These studies investigate the role of uncoupling protein 3 (UCP3) in cardiac energy metabolism, cardiac O(2) consumption (MVO(2)), cardiac efficiency (CE), and mitochondrial uncoupling in high fat (HF)-fed or leptin-deficient mice. UCP3KO and wild-type (WT) mice were fed normal chow or HF diets for 10 weeks. Substrate utilization rates, MVO(2), CE, and mitochondrial uncoupling were measured in perfused working hearts and saponin-permeabilized cardiac fibers, respectively. Similar analyses were performed in hearts of ob/ob mice lacking UCP3 (U3OB mice). HF increased cardiac UCP3 protein. However, fatty acid (FA) oxidation rates were similarly increased by HF diet in WT and UCP3KO mice. By contrast, MVO(2) increased in WT, but not in UCP3KO with HF, leading to increased CE in UCP3KO mice. Consistent with increased CE, mitochondrial coupling was increased in the hearts of HF-fed UCP3KO mice. Unexpectedly, UCP3 deletion in ob/ob mice reduced FA oxidation but had no effect on MVO(2) or CE. In addition, FA-induced mitochondrial uncoupling was similarly enhanced in U3OB compared with ob/ob hearts and was associated with elevated mitochondrial thioesterase-1 protein content. These studies show that although UCP3 may mediate mitochondrial uncoupling and reduced CE after HF feeding, it does not mediate uncoupling in leptin-deficient states.

Early mitochondrial adaptations in skeletal muscle to diet-induced obesity are strain dependent and determine oxidative stress and energy expenditure but not insulin sensitivity.

This study sought to elucidate the relationship between skeletal muscle mitochondrial dysfunction, oxidative stress, and insulin resistance in two mouse models with differential susceptibility to diet-induced obesity. We examined the time course of mitochondrial dysfunction and insulin resistance in obesity-prone C57B and obesity-resistant FVB mouse strains in response to high-fat feeding. After 5 wk, impaired insulin-mediated glucose uptake in skeletal muscle developed in both strains in the absence of any impairment in proximal insulin signaling. Impaired mitochondrial oxidative capacity preceded the development of insulin resistant glucose uptake in C57B mice in concert with increased oxidative stress in skeletal muscle. By contrast, mitochondrial uncoupling in FVB mice, which prevented oxidative stress and increased energy expenditure, did not prevent insulin resistant glucose uptake in skeletal muscle. Preventing oxidative stress in C57B mice treated systemically with an antioxidant normalized skeletal muscle mitochondrial function but failed to normalize glucose tolerance and insulin sensitivity. Furthermore, high fat-fed uncoupling protein 3 knockout mice developed increased oxidative stress that did not worsen glucose tolerance. In the evolution of diet-induced obesity and insulin resistance, initial but divergent strain-dependent mitochondrial adaptations modulate oxidative stress and energy expenditure without influencing the onset of impaired insulin-mediated glucose uptake.

Rnd3/RhoE Is down-regulated in hepatocellular carcinoma and controls cellular invasion.

UNLABELLED: We performed a review of public microarray data that revealed a significant down-regulation of Rnd3 expression in hepatocellular carcinoma (HCC), as compared to nontumor liver. Rnd3/RhoE is an atypical RhoGTPase family member because it is always under its active GTP-bound conformation and not sensitive to classical regulators. Rnd3 down-regulation was validated by quantitative real-time polymerase chain reaction in 120 independent tumors. Moreover, Rnd3 down-expression was confirmed using immunohistochemistry on tumor sections and western blotting on human tumor and cell-line extracts. Rnd3 expression was significantly lower in invasive tumors with satellite nodules. Overexpression and silencing of Rnd3 in Hep3B cells led to decreased and increased three-dimensional cell motility, respectively. The short interfering RNA-mediated down-regulation of Rnd3 expression induced a loss of E-cadherin at cell-cell junctions that was linked to epithelial-mesenchymal transition through the up-regulation of the zinc finger E-box binding homeobox protein, ZEB2, and the down-regulation of miR-200b and miR-200c. Rnd3 knockdown mediated tumor hepatocyte invasion in a matrix-metalloproteinase-independent, and Rac1-dependent manner. CONCLUSION: Rnd3 down-regulation provides an invasive advantage to tumor hepatocytes, suggesting that RND3 might represent a metastasis suppressor gene in HCC.

Knockout of insulin receptors in cardiomyocytes attenuates coronary arterial dysfunction induced by pressure overload.

Ablating insulin receptors in cardiomyocytes causes subendocardial fibrosis and left ventricular (LV) dysfunction after 4 wk of transverse aortic constriction (TAC). To determine whether these maladaptive responses are precipitated by coronary vascular dysfunction, we studied mice with cardiomyocyte-restricted knock out of insulin receptors (CIRKO) and wild-type (WT) TAC mice before the onset of overt LV dysfunction. Two weeks of TAC produced comparable increases (P < 0.05 vs. respective sham) in heart weight/body weight (mg/g) in WT-TAC (8.03 ± 1.14, P < 0.05 vs. respective sham) and CIRKO-TAC (7.76 ± 1.25, P < 0.05 vs. respective sham) vs. WT-sham (5.64 ± 0.11) and CIRKO-sham (4.64 ± 0.10) mice. In addition, 2 wk of TAC were associated with similar LV geometry and function (echocardiography) and interstitial fibrosis (picrosirius red staining) in CIRKO and WT mice. Responses to acetylcholine (ACh), N(G)-monomethyl-L-arginine (l-NMMA), and sodium nitroprusside (SNP) were measured in coronary arteries that were precontracted to achieve ∼70% of maximal tension development using the thromboxane A(2) receptor mimetic U-46619 (∼3 × 10(-6) M). ACh-evoked vasorelaxation was absent in WT-TAC but was present in CIRKO-TAC albeit reduced relative to sham-operated animals. l-NMMA-evoked tension development was similar in vessels from CIRKO-TAC mice but was lower (P < 0.05) in WT-TAC animals vs. the respective sham-operated groups, and SNP-evoked vasorelaxation was similar among all mice. Thus estimates of stimulated and basal endothelial nitric oxide release were better preserved in CIRKO vs. WT mice in response to 2 wk of TAC. These findings indicate that maladaptive LV remodeling previously observed in CIRKO-TAC mice is not precipitated by coronary artery dysfunction, because CIRKO mice exhibit compensatory mechanisms (e.g., increased eNOS transcript and protein) to maintain coronary endothelial function in the setting of pressure overload.

Mammalian target of rapamycin is a critical regulator of cardiac hypertrophy in spontaneously hypertensive rats.

Evidence exists that protein kinase C and the mammalian target of rapamycin are important regulators of cardiac hypertrophy. We examined the contribution of these signaling kinases to cardiac growth in spontaneously hypertensive rats (SHRs). Systolic blood pressure was increased (P<0.001) at 10 weeks in SHRs versus Wistar-Kyoto controls (162+/-3 versus 128+/-1 mm Hg) and was further elevated (P<0.001) at 17 weeks in SHRs (184+/-7 mm Hg). Heart:body weight ratio was not different between groups at 10 weeks but was 22% greater (P<0.01) in SHRs versus Wistar-Kyoto controls at 17 weeks. At 10 weeks, activation of Akt and S6 ribosomal protein was greater (P<0.01) in SHRs but returned to normal by 17 weeks. In contrast, SHRs had protein kinase C activation only at 17 weeks. To determine whether mammalian target of rapamycin regulates the initial development of hypertrophy, rats were treated with rapamycin (2 mg/kg per day IP) or saline vehicle from 13 to 16 weeks of age. Rapamycin inhibited cardiac mammalian target of rapamycin in SHRs, as evidenced by reductions (P<0.001) in phosphorylation of S6 ribosomal protein and eukaryotic translation initiation factor-4E binding protein 1. Rapamycin treatment also reduced (P<0.001) heart weight and hypertrophy by 47% and 53%, respectively, in SHRs in spite of increased (P<0.001) systolic blood pressure versus untreated SHRs (213+/-8 versus 189+/-6 mm Hg). Atrial natriuretic peptide, brain natriuretic peptide, and cardiac function were unchanged between SHRs treated with rapamycin or vehicle. These data show that mammalian target of rapamycin is required for the development of cardiac hypertrophy evoked by rising blood pressure in SHRs.

Impaired insulin signaling accelerates cardiac mitochondrial dysfunction after myocardial infarction.

Diabetes increases mortality and accelerates left ventricular (LV) dysfunction following myocardial infarction (MI). This study sought to determine the impact of impaired myocardial insulin signaling, in the absence of diabetes, on the development of LV dysfunction following MI. Mice with cardiomyocyte-restricted knock out of the insulin receptor (CIRKO) and wildtype (WT) mice were subjected to proximal left coronary artery ligation (MI) and followed for 14 days. Despite equivalent infarct size, mortality was increased in CIRKO-MI vs. WT-MI mice (68% vs. 40%, respectively). In surviving mice, LV ejection fraction and dP/dt were reduced by >40% in CIRKO-MI vs. WT-MI. Relative to shams, isometric developed tension in LV papillary muscles increased in WT-MI but not in CIRKO-MI. Time to peak tension and relaxation times were prolonged in CIRKO-MI vs. WT-MI suggesting impaired, load-independent myocardial contractile function. To elucidate mechanisms for impaired LV contractility, mitochondrial function was examined in permeabilized cardiac fibers. Whereas maximal ADP-stimulated mitochondrial O(2) consumption rates (V(ADP)) with palmitoyl carnitine were unchanged in WT-MI mice relative to sham-operated animals, V(ADP) was significantly reduced in CIRKO-MI (13.17+/-0.94 vs. 9.14+/-0.88 nmol O(2)/min/mgdw, p<0.05). Relative to WT-MI, expression levels of GLUT4, PPAR-alpha, SERCA2, and the FA-Oxidation genes MCAD, LCAD, CPT2 and the electron transfer flavoprotein ETFDH were repressed in CIRKO-MI. Thus reduced insulin action in cardiac myocytes accelerates post-MI LV dysfunction, due in part to a rapid decline in mitochondrial FA oxidative capacity, which combined with limited glucose transport capacity that may reduce substrate utilization and availability.

Contribution of impaired myocardial insulin signaling to mitochondrial dysfunction and oxidative stress in the heart.

BACKGROUND: Diabetes-associated cardiac dysfunction is associated with mitochondrial dysfunction and oxidative stress, which may contribute to left ventricular dysfunction. The contribution of altered myocardial insulin action, independent of associated changes in systemic metabolism, is incompletely understood. The present study tested the hypothesis that perinatal loss of insulin signaling in the heart impairs mitochondrial function. METHODS AND RESULTS: In 8-week-old mice with cardiomyocyte deletion of insulin receptors (CIRKO), inotropic reserves were reduced, and mitochondria manifested respiratory defects for pyruvate that was associated with proportionate reductions in catalytic subunits of pyruvate dehydrogenase. Progressive age-dependent defects in oxygen consumption and ATP synthesis with the substrate glutamate and the fatty acid derivative palmitoyl-carnitine were observed. Mitochondria also were uncoupled when exposed to palmitoyl-carnitine, in part as a result of increased reactive oxygen species production and oxidative stress. Although proteomic and genomic approaches revealed a reduction in subsets of genes and proteins related to oxidative phosphorylation, no reductions in maximal activities of mitochondrial electron transport chain complexes were found. However, a disproportionate reduction in tricarboxylic acid cycle and fatty acid oxidation proteins in mitochondria suggests that defects in fatty acid and pyruvate metabolism and tricarboxylic acid flux may explain the mitochondrial dysfunction observed. CONCLUSIONS: Impaired myocardial insulin signaling promotes oxidative stress and mitochondrial uncoupling, which, together with reduced tricarboxylic acid and fatty acid oxidative capacity, impairs mitochondrial energetics. This study identifies specific contributions of impaired insulin action to mitochondrial dysfunction in the heart.

Mechanisms for increased myocardial fatty acid utilization following short-term high-fat feeding.

AIMS: Diet-induced obesity is associated with increased myocardial fatty acid (FA) utilization, insulin resistance, and cardiac dysfunction. The study was designed to test the hypothesis that impaired glucose utilization accounts for initial changes in FA metabolism. METHODS AND RESULTS: Ten-week-old C57BL6J mice were fed a high-fat diet (HFD, 45% calories from fat) or normal chow (4% calories from fat). Cardiac function and substrate metabolism in isolated working hearts, glucose uptake in isolated cardiomyocytes, mitochondrial function, insulin-stimulated protein kinase B (Akt/PKB) and Akt substrate (AS-160) phosphorylation, glucose transporter 4 (GLUT4) translocation, pyruvate dehydrogenase (PDH) activity, and mRNA levels for metabolic genes were determined after 2 or 5 weeks of HFD. Two weeks of HFD reduced basal rates of glycolysis and glucose oxidation and prevented insulin stimulation of glycolysis in hearts and reduced insulin-stimulated glucose uptake in cardiomyocytes. Insulin-stimulated Akt/PKB and AS-160 phosphorylation were preserved, and PDH activity was unchanged. GLUT4 content was reduced by 55% and GLUT4 translocation was significantly attenuated. HFD increased FA oxidation rates and myocardial oxygen consumption (MVO2), which could not be accounted for by mitochondrial uncoupling or by increased expression of peroxisome proliferator activated receptor-alpha (PPAR-alpha) target genes, which increased only after 5 weeks of HFD. CONCLUSION: Rates of myocardial glucose utilization are altered early in the course of HFD because of reduced GLUT4 content and GLUT4 translocation despite normal insulin signalling to Akt/PKB and AS-160. The reciprocal increase in FA utilization is not due to PPAR-alpha-mediated signalling or mitochondrial uncoupling. Thus, the initial increase in myocardial FA utilization in response to HFD likely results from impaired glucose transport that precedes impaired insulin signalling.

Insulin-like growth factor I receptor signaling is required for exercise-induced cardiac hypertrophy.

The receptors for IGF-I (IGF-IR) and insulin (IR) have been implicated in physiological cardiac growth, but it is unknown whether IGF-IR or IR signaling are critically required. We generated mice with cardiomyocyte-specific knockout of IGF-IR (CIGF1RKO) and compared them with cardiomyocyte-specific insulin receptor knockout (CIRKO) mice in response to 5 wk exercise swim training. Cardiac development was normal in CIGF1RKO mice, but the hypertrophic response to exercise was prevented. In contrast, despite reduced baseline heart size, the hypertrophic response of CIRKO hearts to exercise was preserved. Exercise increased IGF-IR content in control and CIRKO hearts. Akt phosphorylation increased in exercise-trained control and CIRKO hearts and, surprisingly, in CIGF1RKO hearts as well. In exercise-trained control and CIRKO mice, expression of peroxisome proliferator-activated receptor-gamma coactivator-1alpha (PGC-1alpha) and glycogen content were both increased but were unchanged in trained CIGF1RKO mice. Activation of AMP-activated protein kinase (AMPK) and its downstream target eukaryotic elongation factor-2 was increased in exercise-trained CIGF1RKO but not in CIRKO or control hearts. In cultured neonatal rat cardiomyocytes, activation of AMPK with 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICAR) prevented IGF-I/insulin-induced cardiomyocyte hypertrophy. These studies identify an essential role for IGF-IR in mediating physiological cardiomyocyte hypertrophy. IGF-IR deficiency promotes energetic stress in response to exercise, thereby activating AMPK, which leads to phosphorylation of eukaryotic elongation factor-2. These signaling events antagonize Akt signaling, which although necessary for mediating physiological cardiac hypertrophy, is insufficient to promote cardiac hypertrophy in the absence of myocardial IGF-I signaling.

Cardiac hypertrophy caused by peroxisome proliferator- activated receptor-gamma agonist treatment occurs independently of changes in myocardial insulin signaling.

Peroxisome proliferator-activated receptor (PPAR)-gamma ligands are insulin sensitizers, widely used in the treatment of type 2 diabetes. A consistent observation in preclinical species is the development of cardiac hypertrophy after short-term treatment with these agents. The mechanisms for this hypertrophy are incompletely understood. Given the important role of insulin signaling in the regulation of myocardial size, we tested the hypothesis that augmentation of myocardial insulin signaling may play a role in PPAR-gamma ligand-induced cardiac hypertrophy. We treated mice with cardiomyocyte-restricted knockout of insulin receptors (CIRKO) and littermate controls (wild type) with 2-(2-(4-phenoxy-2-propylphenoxy) ethyl) indole-5-acetic acid (COOH), which is a non-thiazolidinedione PPAR-gamma agonist for 2 wk. Two weeks of COOH treatment increased heart weights by 22% in CIRKO mice and 16% in wild type, and induced similar fold increase in the expression of hypertrophic markers such as alpha-skeletal actin, brain natriuretic peptide, and atrial natriuretic peptide in CIRKO and wild-type (WT) hearts. COOH treatment increased plasma volume by 10% in COOH-treated WT and CIRKO mice but did not increase systolic or diastolic blood pressure. Echocardiographic analysis was also consistent with volume overload, as evidenced by increased left ventricular diastolic diameters and cardiac output in COOH-treated CIRKO and WT mice. These data indicate that cardiac hypertrophy after PPAR-gamma agonist treatment can occur in the absence of myocardial insulin signaling and is likely secondary to the hemodynamic consequences of plasma volume expansion.

Mitochondrial energetics in the heart in obesity-related diabetes: direct evidence for increased uncoupled respiration and activation of uncoupling proteins.

OBJECTIVE: In obesity and diabetes, myocardial fatty acid utilization and myocardial oxygen consumption (MVo(2)) are increased, and cardiac efficiency is reduced. Mitochondrial uncoupling has been proposed to contribute to these metabolic abnormalities but has not been directly demonstrated. RESEARCH DESIGN AND METHODS: Oxygen consumption and cardiac function were determined in db/db hearts perfused with glucose or glucose and palmitate. Mitochondrial function was determined in saponin-permeabilized fibers and proton leak kinetics and H(2)O(2) generation determined in isolated mitochondria. RESULTS: db/db hearts exhibited reduced cardiac function and increased MVo(2). Mitochondrial reactive oxygen species (ROS) generation and lipid and protein peroxidation products were increased. Mitochondrial proliferation was increased in db/db hearts, oxidative phosphorylation capacity was impaired, but H(2)O(2) production was increased. Mitochondria from db/db mice exhibited fatty acid-induced mitochondrial uncoupling that is inhibitable by GDP, suggesting that these changes are mediated by uncoupling proteins (UCPs). Mitochondrial uncoupling was not associated with an increase in UCP content, but fatty acid oxidation genes and expression of electron transfer flavoproteins were increased, whereas the content of the F1 alpha-subunit of ATP synthase was reduced. CONCLUSIONS: These data demonstrate that mitochondrial uncoupling in the heart in obesity and diabetes is mediated by activation of UCPs independently of changes in expression levels. This likely occurs on the basis of increased delivery of reducing equivalents from beta-oxidation to the electron transport chain, which coupled with decreased oxidative phosphorylation capacity increases ROS production and lipid peroxidation.

Reduced mitochondrial oxidative capacity and increased mitochondrial uncoupling impair myocardial energetics in obesity.

BACKGROUND: Obesity is a risk factor for cardiovascular disease and is strongly associated with insulin resistance and type 2 diabetes. Recent studies in obese humans and animals demonstrated increased myocardial oxygen consumption (MVO2) and reduced cardiac efficiency (CE); however, the underlying mechanisms remain unclear. The present study was performed to determine whether mitochondrial dysfunction and uncoupling are responsible for reduced cardiac performance and efficiency in ob/ob mice. METHODS AND RESULTS: Cardiac function, MVO2, mitochondrial respiration, and ATP synthesis were measured in 9-week-old ob/ob and control mouse hearts. Contractile function and MVO2 in glucose-perfused ob/ob hearts were similar to controls under basal conditions but were reduced under high workload. Perfusion of ob/ob hearts with glucose and palmitate increased MVO2 and reduced CE by 23% under basal conditions, and CE remained impaired at high workload. In glucose-perfused ob/ob hearts, mitochondrial state 3 respirations were reduced but ATP/O ratios were unchanged. In contrast, state 3 respiration rates were similar in ob/ob and control mitochondria from hearts perfused with palmitate and glucose, but ATP synthesis rates and ATP/O ratios were significantly reduced in ob/ob, which suggests increased mitochondrial uncoupling. Pyruvate dehydrogenase activity and protein levels of complexes I, III, and V were reduced in obese mice. CONCLUSIONS: These data indicate that reduced mitochondrial oxidative capacity may contribute to cardiac dysfunction in ob/ob mice. Moreover, fatty acid but not glucose-induced mitochondrial uncoupling reduces CE in obese mice by limiting ATP production and increasing MVO2.

In vitro prevention of cyclosporin-induced cell contraction by mycophenolic acid.

Nephrotoxicity is a major side-effect of cyclosporin A (CsA), which induces a vasoconstrictive response in vascular smooth muscle and mesangial cells. Mycophenolic acid (MPA) is used in combination with low-dose CsA to reduce nephrotoxicity. We previously demonstrated that MPA affected mesangial cell contractile response to angiotensin II or KCl. Aims of the present study were to evaluate if MPA can prevent CsA-induced contraction of human mesangial and aortic smooth muscle cells (ASMC). Using a morphoquantitative approach, we evidenced that pretreatment with MPA (1 microM) prevented the reduction of cell area induced by CsA within 30 min in both cell types. We then compared the expression of three main cytoskeleton proteins: tubulin, alpha-smooth actin (SMA) and basic calponin, in ASMC and in mesangial cells treated with MPA and/or CsA. CsA alone did not significantly change the expression level of these proteins neither in mesangial cells nor in ASMC. MPA decreased the expression level of tubulin in both mesangial cells and ASMC. Surprisingly, MPA, which stimulated SMA and calponin expression in mesangial cells, exerted an inhibitory effect on both contractile protein expression in ASMC. In conclusion, our results evidenced opposite effects of MPA on calponin and SMA protein expression in ASMC and in mesangial cells, despite similar antiproliferative properties, suggesting that sarcomeric protein expression is controlled by different intracellular mechanisms in mesangial and smooth muscle cells. However, MPA interferes in both cell types with the constrictive properties CsA, which may partially explain the protective effects of MPA against CsA nephrotoxicity.