Interventions against obesity through increased lipolysis in adipose tissue

Obesity is a disease of excess adiposity affecting> 17% of men and >20% of women in Britain. Clinically, it is defined by a Body Mass Index (BMI, kg/m2) of 2:30. Obesity is a confounding factor that promotes insulin resistance, hyperinsulinaemia and type 2 diabetes. Type 2 diabetes accounts for >90% of all cases of diabetes, with a prevalence of 2-6% of adults in most western societies, a majority of which are overweight or obese. Weight loss in obese patients reduces the risk of developing diabetes by >50%. This thesis has investigated the first part of a two-stage therapeutic intervention against obesity in which adipose tissue lipolysis will be combined with increased energy expenditure: the approach is also designed to consider agents that will benefit glycaemic control in coexistent obesity and diabetes by improving insulin sensitivity. Rodent and human in vitro models of adipocyte biology and skeletal muscle have been developed, characterised and evaluated. They include isolated epididymal and parametrial adipocytes of lean and obese diabetic ob/ob mice, cultured 3T3-Ll preadipocytes, isolated human omental and subcutaneous adipocytes and rat L6 cultured muscle cells. Compounds investigated for anti-obesity and anti-diabetic properties include M2 (sibutramine metabolite), 3-guanidinopropionic acid and mazindol. In vivo studies were undertaken to investigate these compounds further in lean and ob/ob mice. In vivo studies indicated that M2 and 3-guanidinopropionic acid reduced body weight gain in ob/ob mice. The three compounds increased lipolysis in adipocytes isolated from lean and ob/ob mice and human adipose depots. The direct action of these compounds was mediated via a pathway involving the f3 adrenoceptors and components of the lipolytic signalling pathway, including protein kinase A and p38 MAP kinase. In addition, M2 and mazindol were capable of increasing glucose uptake into insulin sensitive tissues. M2 and mazindol can act directly on adipose tissue and skeletal muscle to increase glucose uptake via a pathway involving new protein synthesis and activation of the glucose transporters. The M2-stimulated pathway is activated by the conversion of phosphatidylinositol bisphosphate to phosphatidylinositol trisphosphate by phosphatidylinositol 3-kinase. Thus, M2, mazindol and 3-GPA showed pharmacodynamic properties which suggested they might be potential therapeutic treatments for obesity and diabetes.

Autocrine activity of soluble Flt-1 controls endothelial cell function and angiogenesis

Background - The negative feedback system is an important physiological regulatory mechanism controlling angiogenesis. Soluble vascular endothelial growth factor (VEGF) receptor-1 (sFlt-1), acts as a potent endogenous soluble inhibitor of VEGF- and placenta growth factor (PlGF)-mediated biological function and can also form dominant-negative complexes with competent full-length VEGF receptors. Methods and results - Systemic overexpression of VEGF-A in mice resulted in significantly elevated circulating sFlt-1. In addition, stimulation of human umbilical vein endothelial cells (HUVEC) with VEGF-A, induced a five-fold increase in sFlt-1 mRNA, a time-dependent significant increase in the release of sFlt-1 into the culture medium and activation of the flt-1 gene promoter. This response was dependent on VEGF receptor-2 (VEGFR-2) and phosphoinositide-3'-kinase signalling. siRNA-mediated knockdown of sFlt-1 in HUVEC stimulated the activation of endothelial nitric oxide synthase, increased basal and VEGF-induced cell migration and enhanced endothelial tube formation on growth factor reduced Matrigel. In contrast, adenoviral overexpression of sFlt-1 suppressed phosphorylation of VEGFR-2 at tyrosine 951 and ERK-1/-2 MAPK and reduced HUVEC proliferation. Preeclampsia is associated with elevated placental and systemic sFlt-1. Phosphorylation of VEGFR-2 tyrosine 951 was greatly reduced in placenta from preeclamptic patients compared to gestationally-matched normal placenta. Conclusion - These results show that endothelial sFlt-1 expression is regulated by VEGF and acts as an autocrine regulator of endothelial cell function.

Loss of Akt activity increases circulating soluble endoglin release in preeclampsia:identification of inter-dependency between Akt-1 and heme oxygenase-1

Aims - Endothelial dysfunction is a hallmark of preeclampsia. Desensitization of the phosphoinositide 3-kinase (PI3K)/Akt pathway underlies endothelial dysfunction and haeme oxygenase-1 (HO-1) is decreased in preeclampsia. To identify therapeutic targets, we sought to assess whether these two regulators act to suppress soluble endoglin (sEng), an antagonist of transforming growth factor-ß (TGF-ß) signalling, which is known to be elevated in preeclampsia. Methods and results - Vascular endothelial growth factor-A (VEGF-A), fibroblast growth factor (FGF-2), angiopoietin-1 (Ang-1), and insulin, which all activate the PI3K/Akt pathway, inhibited the release of sEng from endothelial cells. Inhibition of the PI3K/Akt pathway, by overexpression of phosphatase and tensin homolog (PTEN) or a dominant-negative isoform of Akt (Aktdn) induced sEng release from endothelial cells and prevented the inhibitory effect of VEGF-A. Conversely, overexpression of a constitutively active Akt (Aktmyr) inhibited PTEN and cytokine-induced sEng release. Systemic delivery of Aktmyr to mice significantly reduced circulating sEng, whereas Aktdn promoted sEng release. Phosphorylation of Akt was reduced in preeclamptic placenta and this correlated with the elevated level of circulating sEng. Knock-down of Akt using siRNA prevented HO-1-mediated inhibition of sEng release and reduced HO-1 expression. Furthermore, HO-1 null mice have reduced phosphorylated Akt in their organs and overexpression of Aktmyr failed to suppress the elevated levels of sEng detected in HO-1 null mice, indicating that HO-1 is required for the Akt-mediated inhibition of sEng. Conclusion - The loss of PI3K/Akt and/or HO-1 activity promotes sEng release and positive manipulation of these pathways offers a strategy to circumvent endothelial dysfunction.

Direct evidence for endothelial vascular endothelial growth factor receptor-1 function in nitric oxide-mediated angiogenesis

Vascular endothelial growth factor-A (VEGF) is critical for angiogenesis but fails to induce neovascularization in ischemic tissue lesions in mice lacking endothelial nitric oxide synthase (eNOS). VEGF receptor-2 (VEGFR-2) is critical for angiogenesis, although little is known about the precise role of endothelial VEGFR-1 and its downstream effectors in this process. Here we have used a chimeric receptor approach in which the extracellular domain of the epidermal growth factor receptor was substituted for that of VEGFR-1 (EGLT) or VEGFR-2 (EGDR) and transduced into primary cultures of human umbilical vein endothelial cells (HUVECs) using a retroviral system. Activation of HUVECs expressing EGLT or EGDR induced rapid phosphorylation of eNOS at Ser1177, release of NO, and formation of capillary networks, similar to VEGF. Activation of eNOS by VEGFR-1 was dependent on Tyr794 and was mediated via phosphatidylinositol 3-kinase, whereas VEGFR-2 Tyr951 was involved in eNOS activation via phospholipase Cgamma1. Consistent with these findings, the VEGFR-1-specific ligand placenta growth factor-1 activated phosphatidylinositol 3-kinase and VEGF-E, which is selective for VEGFR-2-activated phospholipase Cgamma1. Both VEGFR-1 and VEGFR-2 signal pathways converged on Akt, as dominant-negative Akt inhibited the NO release and in vitro tube formation induced following activation of EGLT and EGDR. The identification Tyr794 of VEGFR-1 as a key residue in this process provides direct evidence of endothelial VEGFR-1 in NO-driven in vitro angiogenesis. These studies provide new sites of modulation in VEGF-mediated vascular morphogenesis and highlight new therapeutic targets for management of vascular diseases.

Vascular endothelial growth factor promotes physical wound repair and is anti-apoptotic in primary distal lung epithelial and A549 cells

Objective: There is evidence to suggest a beneficial role for growth factors, including vascular endothelial growth factor (VEGF), in tissue repair and proliferation after injury within the lung. Whether this effect is mediated predominantly by actions on endothelial cells or epithelial cells is unknown. This study tested the hypothesis that VEGF acts as an autocrine trophic factor for human adult alveolar epithelial cells and that under situations of pro-apoptotic stress, VEGF reduces cell death. Design: In vitro cell culture study looking at the effects of 0.03% H2O2 on both A549 and primary distal lung epithelial cells.Measurement and Main Results: Primary adult human distal lung epithelial cells express both the soluble and membrane-associated VEGF isoforms and VEGF receptors 1 and 2. At physiologically relevant doses, soluble VEGF isoforms stimulate wound repair and have a proliferative action. Specific receptor ligands confirmed that this effect was mediated by VEGF receptor 1. In addition to proliferation, we demonstrate that VEGF reduces A549 and distal lung epithelial cell apoptosis when administered after 0.03% H2O2 injury. This effect occurs due to reduced caspase-3 activation and is phosphatidylinositol 3′–kinase dependent. Conclusion: In addition to its known effects on endothelial cells, VEGF acts as a growth and anti-apoptotic factor on alveolar epithelial cells. VEGF treatment may have potential as a rescue therapy for diseases associated with alveolar epithelial damage such as acute respiratory distress syndrome.

Angiopoietin-1 induces migration of monocytes in a tie-2 and integrin-independent manner

Angiopoietin-1 (Ang-1) is an angiogenic growth factor that activates Tie-2 and integrins to promote vessel wall remodeling. The recent finding of the potential proatherogenic effects of Ang-1 prompted us to investigate whether Ang-1 promotes monocyte chemotaxis, endothelial binding, and transendothelial migration, key events in the progression of atherosclerosis. Here, we show that Ang-1 induces chemotaxis of monocytes in a manner that is independent of Tie-2 and integrin binding but dependent on phosphoinositide 3-kinase and heparin. In addition, Ang-1 promoted phosphoinositide 3-kinase-dependent binding of monocytes to endothelial monolayers and stimulated transendothelial migration. Fluorescence-activated cell sorting analysis showed that exogenous Ang-1 adheres directly to monocytes as well as to human umbilical endothelial cells, but neither Tie-2 mRNA nor protein were expressed by primary monocytes. Although Ang-1 binding to human umbilical endothelial cells was partially Tie-2 and integrin dependent, Ang-1 binding to monocytes was independent of these factors. Finally, preincubation of monocytes with soluble heparin abrogated Ang-1 binding to monocytes and migration, and partially prevented Ang-1 binding to human umbilical endothelial cells. In summary, Ang-1 induces chemotaxis of monocytes by a mechanism that is dependent on phosphoinositide 3-kinase and heparin but independent of Tie-2 and integrins. The ability of Ang-1 to recruit monocytes suggests it may play a role in inflammatory angiogenesis and may promote atherosclerosis.

Evaluating the evidence for targeting FOXO3a in breast cancer:a systematic review

Background: Tumour cells show greater dependency on glycolysis so providing a sufficient and rapid energy supply for fast growth. In many breast cancers, estrogen, progesterone and epidermal growth factor receptor-positive cells proliferate in response to growth factors and growth factor antagonists are a mainstay of treatment. However, triple negative breast cancer (TNBC) cells lack receptor expression, are frequently more aggressive and are resistant to growth factor inhibition. Downstream of growth factor receptors, signal transduction proceeds via phosphatidylinositol 3-kinase (PI3k), Akt and FOXO3a inhibition, the latter being partly responsible for coordinated increases in glycolysis and apoptosis resistance. FOXO3a may be an attractive therapeutic target for TNBC. Therefore we have undertaken a systematic review of FOXO3a as a target for breast cancer therapeutics. Methods: Articles from NCBI were retrieved systematically when reporting primary data about FOXO3a expression in breast cancer cells after cytotoxic drug treatment. Results: Increased FOXO3a expression is common following cytotoxic drug treatment and is associated with apoptosis and cell cycle arrest. There is some evidence that metabolic enzyme expression is also altered and that this effect is also elicited in TNBC cells. FOXO3a expression serves as a positive prognostic marker, especially in estrogen (ER) receptor positive cells. Discussion: FOXO3a is upregulated by a number of receptor-dependent and -independent anti-cancer drugs and associates with apoptosis. The identification of microRNA that regulate FOXO3a directly suggest that it offers a tangible therapeutic target that merits wider evaluation.

Le glucagon-like peptide-I : un facteur de croissance et une hormone anti-apoptotique pour la cellule pancréatique[bêta] : étude de la transduction du signal

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

Rôle de la protéine adaptatrice APS dans les voies de signalisation du récepteur [bêta] du PDGF

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Dual modulation of cell survival and cell death by beta(2)-adrenergic signaling in adult mouse cardiac myocytes.

The goal of this study was to determine whether beta(1)-adrenergic receptor (AR) and beta(2)-AR differ in regulating cardiomyocyte survival and apoptosis and, if so, to explore underlying mechanisms. One potential mechanism is that cardiac beta(2)-AR can activate both G(s) and G(i) proteins, whereas cardiac beta(1)-AR couples only to G(s). To avoid complicated crosstalk between beta-AR subtypes, we expressed beta(1)-AR or beta(2)-AR individually in adult beta(1)/beta(2)-AR double knockout mouse cardiac myocytes by using adenoviral gene transfer. Stimulation of beta(1)-AR, but not beta(2)-AR, markedly induced myocyte apoptosis, as indicated by increased terminal deoxynucleotidyltransferase-mediated UTP end labeling or Hoechst staining positive cells and DNA fragmentation. In contrast, beta(2)-AR (but not beta(1)-AR) stimulation elevated the activity of Akt, a powerful survival signal; this effect was fully abolished by inhibiting G(i), G(beta gamma), or phosphoinositide 3 kinase (PI3K) with pertussis toxin, beta ARK-ct (a peptide inhibitor of G(beta gamma)), or LY294002, respectively. This indicates that beta(2)-AR activates Akt via a G(i)-G(beta gamma)-PI3K pathway. More importantly, inhibition of the G(i)-G(beta gamma)-PI3K-Akt pathway converts beta(2)-AR signaling from survival to apoptotic. Thus, stimulation of a single class of receptors, beta(2)-ARs, elicits concurrent apoptotic and survival signals in cardiac myocytes. The survival effect appears to predominate and is mediated by the G(i)-G(beta gamma)-PI3K-Akt signaling pathway.

Le glucagon-like peptide-I : un facteur de croissance et une hormone anti-apoptotique pour la cellule pancréatique[bêta] : étude de la transduction du signal

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

Rôle de la protéine adaptatrice APS dans les voies de signalisation du récepteur [bêta] du PDGF

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Antioxidants attenuate hyperglycaemia-mediated brain endothelial cell dysfunction and blood–brain barrier hyperpermeability

Aims: Hyperglycaemia (HG), in stroke patients, is associated with worse neurological outcome by compromising endothelial cell function and the blood–brain barrier (BBB) integrity. We have studied the contribution of HG-mediated generation of oxidative stress to these pathologies and examined whether antioxidants as well as normalization of glucose levels following hyperglycaemic insult reverse these phenomena. Methods: Human brain microvascular endothelial cell (HBMEC) and human astrocyte co-cultures were used to simulate the human BBB. The integrity of the BBB was measured by transendothelial electrical resistance using STX electrodes and an EVOM resistance meter, while enzyme activities were measured by specific spectrophotometric assays. Results: After 5 days of hyperglycaemic insult, there was a significant increase in BBB permeability that was reversed by glucose normalization. Co-treatment of cells with HG and a number of antioxidants including vitamin C, free radical scavengers and antioxidant enzymes including catalase and superoxide dismutase mimetics attenuated the detrimental effects of HG. Inhibition of p38 mitogen-activated protein kinase (p38MAPK) and protein kinase C but not phosphoinositide 3 kinase (PI3 kinase) also reversed HG-induced BBB hyperpermeability. In HBMEC, HG enhanced pro-oxidant (NAD(P)H oxidase) enzyme activity and expression that were normalized by reverting to normoglycaemia. Conclusions: HG impairs brain microvascular endothelial function through involvements of oxidative stress and several signal transduction pathways.

Mir-190b negatively contributes to the Trypanosoma cruzi -infected cell survival by repressing PTEN protein expression

Chagas disease, which is caused by the intracellular protozoan Trypanosoma cruzi , is a serious health problem in Latin America. The heart is one of the major organs affected by this parasitic infection. The pathogenesis of tissue remodelling, particularly regarding cardiomyocyte behaviour after parasite infection, and the molecular mechanisms that occur immediately following parasite entry into host cells are not yet completely understood. Previous studies have reported that the establishment of parasitism is connected to the activation of the phosphatidylinositol- 3 kinase (PI3K), which controls important steps in cellular metabolism by regulating the production of the second messenger phosphatidylinositol-3,4,5-trisphosphate. Particularly, the tumour suppressor PTEN is a negative regulator of PI3K signalling. However, mechanistic details of the modulatory activity of PTEN on Chagas disease have not been elucidated. To address this question, H9c2 cells were infected with T. cruzi Berenice 62 strain and the expression of a specific set of microRNAs (miRNAs) were investigated. Our cellular model demonstrated that miRNA-190b is correlated to the decrease of cellular viability rates by negatively modulating PTEN protein expression in T. cruzi-infected cells.

Heterodimerisation between VEGFR-1 and VEGFR-2 and not the homodimers of VEGFR-1 inhibit VEGFR-2 activity

Vascular endothelial growth factor (VEGF) signaling is tightly regulated by specific VEGF receptors (VEGF-R). Recently, we identified heterodimerisation between VEGFR-1 and VEGFR-2 (VEGFR1–2) to regulate VEGFR-2 function. However, both the mechanism of action and the relationship with VEGFR-1 homodimers remain unknown. The current study shows that activation of VEGFR1–2, but not VEGFR-1 homodimers, inhibits VEGFR-2 receptor phosphorylation under VEGF stimulation in human endothelial cells. Furthermore, inhibition of phosphatidylinositol 3-kinase (PI3K) increases VEGFR-2 phosphorylation under VEGF stimulation. More importantly, inhibition of PI3K pathway abolishes the VEGFR1–2 mediated inhibition of VEGFR-2 phosphorylation. We further demonstrate that inhibition of PI3K pathway promotes capillary tube formation. Finally, the inhibition of PI3K abrogates the inhibition of in vitro angiogenesis mediated by VEGFR1–2 heterodimers. These findings demonstrate that VEGFR1–2 heterodimers and not VEGFR-1 homodimers inhibit VEGF-VEGFR-2 signaling by suppressing VEGFR-2 phosphorylation via PI3K pathway.