Type 2 diabetes mellitus is a metabolic disease that affects one in ten people in the United States. It is caused by a combination of genetics and lifestyle factors. Disease progression begins with insulin resistance in peripheral tissues followed by pancreatic beta-cell failure. The mechanisms behind disease progression are not completely understood. PH domain leucine rich repeat protein phosphatase 1 (PHLPP1) is a known regulator of Akt and other members of the AGC kinase family. Akt has been established to play a role in numerous metabolic signaling pathways, including insulin action. It is hypothesized that as a regulator of Akt, PHLPP1 would have an important function in glucose homeostasis. Glucose tolerance tests performed on 8-week old Phlpp1-/- mice revealed no significant difference in glucose tolerance compared to wild type, however these mice did exhibit increased fasting blood glucose levels. Glucose tolerance tests were repeated at 20 weeks on the same mice and, interestingly, they displayed impaired glucose tolerance compared to wild type. Insulin tolerance tests showed that 8-week old mice have increased insulin sensitivity, however, the 20-week old mice were insulin-resistant compared to control animals. The 20-week old knockout mice also had significantly higher fasting blood glucose levels compared to 8-week old mice. To determine if the increased fasting blood glucose levels are due to increased hepatic glucose output, pyruvate tolerance tests were performed on both the 8 & 20 week old mice. Old mice displayed significantly increased hepatic glucose production compared to wild type. EchoMRI done on 24-week old mice showed significantly increased fat mass and decreased lean mass in the Phlpp1-/- mice compared to wild type littermates. Western blot analysis of liver samples from 32 week old Phlpp1-/- mice indicates loss of Akt signaling accompanied by a decrease in IRS2 protein levels, a common indicator of insulin resistance. These data suggest that Phlpp1-/- mice mimic the development of type 2 diabetes in humans, and provide a unique animal model to study the progression of type 2 diabetes and diabetes-associated complications.

Résumé: La prostaglandine D? (PGD?) est un médiateur lipidique qui active directement deux récepteurs spécifiques, DP et CRTH2, régulant ainsi des processus inflammatoires, immunitaires et apoptotiques. Les ostéoclastes (OC) sont de larges cellules multinucléées participant au métabolisme et remodelage de l’os, ainsi qu’à la réparation de fracture osseuse. Nos travaux ont mis en évidence l’expression des récepteurs DP et CRTH2 chez des OCs humains. Cependant, les effets de la PGD? sur l’apoptose des OCs sont inconnus. L’objectif de la présente étude a été de déterminer si la PGD? induit l’apoptose et les mécanismes qui en découlent dans les OC humains. Les OCs humains différenciés ont été traités avec la PGD?, les agonistes et antagonistes de ses récepteurs. Le traitement des OCs avec la PGD?, en présence de naproxène, qui permet d’inhiber la production endogène de prostaglandines, augmente de façon dépendante de la dose et en fonction du temps le pourcentage d'OCs apoptotiques. Ceci a également été observé lors du traitement des OCs avec l’agoniste spécifique DK-PGD? du récepteur CRTH2, mais pas avec le traitement du composé BW 245C, antagoniste du récepteur DP. En absence de naproxène, l’antagoniste CAY10471 du récepteur CRTH2 réduit le taux d’apoptose des OCs tandis que le composé BW A868C, antagoniste du récepteur DP, n'a aucun effet. L'apoptose des OCs par la PGD? via CRTH2 est associée à l’activation de la caspase-9, et non pas la caspase-8, ce qui entraîne le clivage de la caspase-3. Afin de déterminer plus précisément les mécanismes menant à ces résultats, les OCs ont été traités avec les inhibiteurs de MEK-1/2, PI3K et IKK2/NF-?B. Le traitement des OCs avec la PGD? et l’agoniste de CRTH2 diminue la phosphorylation des protéines ERK1/2 et Akt, tandis que la phosphorylation de ?-arrestine-1 est augmentée. Par ailleurs, les niveaux de phosphorylation d'ERK1/2 et Akt ont été augmentés alors que le taux de protéines ?-arrestine-1 phosphorylees a été diminué par l’antagoniste de CRTH2. En outre, le traitement des OCs avec l’inhibiteur de MEK-1/2 augmente l’apoptose des OCs induite par PGD? et l’agoniste de CRTH2. Cependant, l’antagoniste de CRTH2 diminue l'activité de la caspase-3 induite par l’inhibiteur de MEK1/2. Le traitement des OCs avec l’inhibiteur de la PI3K diminue la phosphorylation d’ERK l/2, tandis que la phosphorylation d'ERK1/2 augmentée par l’antagoniste de CRTH2 a été atténuée par l’inhibiteur de PI3K. Les agonistes et antagonistes du récepteurs DP n'ont pas d’effet sur la phosphorylation d'ERK1/2, Akt, ?-arrestine-1 ni sur l’activité de la caspase-3 chez les OCs. Le traitement des OCs avec PGD? et les ligands de ses récepteurs ne modifie pas la phosphorylation de Re1A/p65. De plus, l’activité de la caspase-3 n'est pas altérée dans les OCs traités avec l’inhibiteur d’IKK2. En conclusion, PGD?, en se liant à CRTH2, induit l’apoptose des OCs via la voie apoptotique intrinsèque qui est associée à la régulation des voies de signalisation des protéines ?-arrestine-1, ERK1/2, et Akt, mais pas celle du IKK2/NF-?B. // Abstract: Prostaglandin D2 (PGD2) is a lipid mediator that directly activates two specific receptors, DP and CRTH2, thereby regulating inflammation, immune response and apoptosis. Osteoclasts (OCs) are large multinucleated cells that participate in bone metabolism, remodeling, and fracture repair. Our previous data show the expression of DP and CRTH2 in human OCs. However, it is unknown whether PGD2 affects OC apoptosis. The objective of the thesis was to determine whether PGD2 induces human OC apoptosis and the underlying mechanisms implicated in this effect. The differentiated human OCs were treated with PGD2, and its receptors agonists/antagonists. Treatment with PGD2 in the presence of naproxen to inhibit endogenous prostaglandins production increased OC apoptosis in a dose- and time-dependent manner, as did the specific CRTH2 agonist compound DK-PGD2 but not the DP agonist compound 13W 245C. In the absence of naproxen, the CRTH2 antagonist compound CAY 10471 reduced OC apoptosis whereas the DP antagonist BW A868C had no such effect. PGD2/CRTH2-induced OC apoptosis was associated with the activation of caspase-9 (an intrinsic apoptosis pathway-initiator caspase), but not caspase-8 (an extrinsic apoptosis pathway-initiator caspase), leading to caspase-3 cleavage. To further determine the mechanisms underlying these findings, human OCs were treated with the inhibitors of MEK-1/2, P13K and IKK2/NF-KB. Treatments with PGD2 and a CRTH2 agonist decreased ERKI/2 and Akt phosphorylation, whereas both treatments increased 13-arrestin- I phosphorylation. Both ERK1/2 and Akt phosphorylation were augmented, whereas the phosphorylated 13-arrestin-1 was reduced by a CRTH2 antagonist. Furthermore, treatment of OCs with a MEK-1/2 inhibitor increased OC apoptosis induced by PGD2 and by a CRTH2 agonist. However, a CRTH2 antagonist diminished the MEK- I /2 inhibitor-induced increase in caspase-3 activity. In addition, treatment of OCs with a PI3K inhibitor decreased ERK I /2 phosphorylation, whereas increased ERK1/2 phosphorylation by CRTH2 antagonist was attenuated by a P13K inhibitor. Both DP receptor agonist and antagonist did not affect either Akt, ERK1/2, 13-arrestin-1 phosphorylation or a specific MEK-1/2 inhibitor-induced increase in caspase-3 activity in OCs. Treatment of OCs with PGD2 and its receptor ligands did not alter ReIA/p65 phosphorylation (ser536). Moreover, the caspase-3 activity was not altered in OCs treated with an IKK2/NF-KB inhibitor. In summary, PGD2 induces human OC apoptosis through a CRTH2-dependent intrinsic apoptosis pathway, which is associated with regulation of the 13-affestin-1, ERK1/2, and Akt, but not with 1KK2/NF-KB, signaling pathways. [symboles non conformes]

The integrin family of cell surface receptors is evolutionary conserved and found in all multicellular animals. In humans 8-alpha and 18-beta integrins are non-covalently associated into 24 dimers. Integrins mediate cell-extracellular matrix and cell-cell interactions and participate in cell signalling. This ideally places integrins to regulate vital processes such as cell adhesion, migration, differentiation and cytoskeleton dynamics. Integrins also play a fundamental role in regulating cell survival and anoikis. In this thesis molecular mechanisms employed by integrins to induce signal transduction, independently or through crosstalk with other receptors, were characterised. Rictor-mTOR (mTORC2) was required for Akt Ser473 phosphorylation in response to β1 integrin-mediated adhesion as well as EGF-, PDGF- or LPA-stimulation of MCF7 cells. ILK and PAK were dispensable for Akt Ser473 phosphorylation upon β1 integrin-engagement or EGF-stimulation. PAK was needed when this phosphorylation was induced by PDGF or LPA. β1 integrin-promoted cell survival during serum starvation conditions was mTORC2 dependent, indicating the importance of Akt Ser473 phosphorylation. mTORC2 was also required for Akt Ser473 phosphorylation induced upon heparanase treatment of cells. Heparanase preferred PI3K catalytic subunit p110α for the upstream lipid phosphorylation required for Akt activation. Interaction between this subunit and Ras was needed for optimal Akt phosphorylation upon heparanase exposure. Cell adhesion strongly promoted heparanase signalling, which was more efficient in β1 integrin-expressing fibroblasts compared to cells lacking this subunit. The cooperative signalling between integrins and heparanase involved FAK and PYK2 since simultaneous silencing of these kinases suppressed heparanase-triggered Akt activation. Furthermore, the resistance of cells to apoptosis induced by H2O2 or serum starvation was promoted by heparanase.  Integrin stimulation by adhesion or cyclic stretching showed divergent downstream signalling responses. Cell attachment on integrin-specific ligands lead to robust phosphorylation of several intracellular integrin-effectors, e.g. p130CAS, FAK, Akt and ERK 1/2. However, mechanical cell stretching only triggered prominent phosphorylation of ERK 1/2. Signalling induced at early stages of integrin-mediated cell adhesion occurred independently of intracellular contraction. Reactive oxygen species (ROS) generated during adhesion and cell stretching influenced integrin signalling. Inhibition of mitochondrial ROS production blocked adhesion-induced Akt phosphorylation. In contrast, stretch-induced ERK 1/2 phosphorylation was elevated when extracellular ROS was scavenged. These results indicate that the two types of integrin stimuli generate signals by different mechanisms.

Bradykinin (BK) acts through eNOS to reduce MAPK-mediated feedback inhibition of insulin signalling. Preliminary data suggest that the sGC-cGMP-PKG pathway, a prominent NO target, is involved. Our present study aimed to support the role of this pathway with atrial natriuretic peptide (ANP), which uses a receptor associated GC (NPR-A) to generate cGMP. We found that treating adipocytes with ANP mimicked BK effects on insulin-stimulated glucose uptake, Tyr-IRS-1 and Akt/PKB phosphorylation, as well as JNK and ERK1/2 inhibition. These outcomes depended on GC-cGMP-PKG signalling since A71915 (NPR-A antagonist), and KT-5823 (PKG inhibitor), completely abrogated them, while zaprinast (phosphodiesterase inhibitor), prolonged ANP actions. Furthermore, decreased MAPK phosphorylation was independent of upstream kinase activity, suggesting that MAPK phosphatases may be involved. These data indicate that BK and ANP act through the GC-cGMP-PKG pathway to potentiate insulin signalling via attenuated feedback inhibition. Stimulating the GC-cGMP-PKG pathway may, therefore, be a promising therapy for T2DM.

Muscle may experience hypoglycemia during ischemia or insulin infusion. During severe hypoglycemia energy production is blocked and an increase in AMP:ATP activates the energy sensor and putative insulin-sensitizer AMP-dependent protein kinase (AMPK). AMPK promotes energy conservation and survival by shutting down anabolism and activating catabolic pathways. We investigated the molecular mechanism of a unique glucose stress defense pathway involving AMPK-dependent, insulin-independent activation of the insulin signaling pathway. Results from my work showed that the central insulin signaling pathway is rapidly activated when cardiac and skeletal myocytes are subjected to conditions of glucose starvation. The effect occurred independently of insulin receptor ligands (insulin and IGF-1). There was a >10-fold increase in the activity of Akt as determined by phosphorylation on both Thr308 and Ser473. Phosphorylation of glycogen synthase 3 beta (GSK3b) increased in parallel, but phosphorylation of ribosomal 70S subunit-S6 protein kinase (S6K) and the mammalian target of rapamycin complex 1 (mTORC1) decreased. We identified AMPK as an intermediate in this signaling network; AMPK was activated by glucose starvation and many of the effects were mimicked by the AMPK-selective activator aminoimidazole carboxamide ribonucleotide (AICAR) and blocked by AMPK inhibitors. Glucose starvation increased the phosphorylation on IRS-1 on Ser789, but phosphomimetics revealed that this conferred negative regulation. Glucose starvation enhanced tyrosine phosphorylation of IRS-1 and the insulin receptor, effects that were blocked by AMPK inhibition and mimicked by AICAR. In vitro kinase assays using purified proteins confirmed that the insulin receptor is a direct target of AMPK. Insulin receptor kinase activity was essential for cardiac myocytes to survive gluose starvation as inhibition of the IR led to increased cell death in glucose-starved myocytes. Selective activation of mTORC2 by glucose starvation to increase Akt-Ser473 phosphorylation was dependent on the presence of rictor. SIN1 also seemed to be instrumental in the activation of mTORC2 as its levels and binding to rictor increased under glucose starvation. AMPK-mediated activation of the insulin signaling pathway conferred significant protection against the stresses of glucose starvation. Glucose starvation promoted energy conservation, augmented glucose uptake and enhanced insulin sensitivity in an AMPK- and Akt-dependent manner. My results describe a novel ligand-independent and AMPK-dependent activation of the insulin signaling pathway via direct phosphorylation and activation of the IR followed by activation of PI3K and Akt. These results may be relevant in conditions of myocardial ischemia superimposed with type 2 diabetes where AMPK could directly modify the IR to promote cell survival and confer protection.

The Snail1 transcriptional repressor is a key factor responsible in triggering epithelial to mesenchymal transition. Although Snail1 is widely expressed in early development, it is limited in adult animals to a subset of mesenchymal cells where it has a largely unknown function. In this project we have demonstrated that Snail1 is required to maintain mesenchymal stem cells (MSCs). This effect is associated to the responsiveness to TGF-[beta]1 which showed a strong Snail1 dependence. Snail1-depletion in conditional knock-out adult animals caused a significant decrease in the number of bone marrow-derived MSCs. In culture, Snail1-deficient MSCs prematurely differentiated to osteoblasts or adipocytes and, in contrast to controls, were resistant to the TGF-[beta]1-induced differentiation block. TGF-[beta]1 was unable to up-regulate most of its targets in Snail1 KO MSCs, an effect that was related, but not limited, to defective PTEN repression and Akt activation. Correspondingly, an analysis of human sarcomas also showed enhanced expression of Snail1 in undifferentiated tumors, which was strongly associated with high expression of TGF-[beta] and poor outcome. These results not only demonstrate a new role for Snail1 in TGF-[beta] response and MSC maintenance but also suggest the involvement of MSCs in sarcoma generation. / El repressor transcripcional Snail1 ha estat descrit principalment com el responsable de la inducció de la transició epiteli mesènquima. Encara que Snail1 s’expressa durant les etapes més primerenques del desenvolupament embrionari, la seva expressió en adults es veu limitada en un conjunt de cèl•lules mesenquimals sense saber-se la seva funció. En aquest projecte hem demostrat que Snail1 es requereix per mantenir el fenotip més indiferenciat de les cèl•lules mare del mesènquima. Aquesta funció la fa en part, per la capacitat de resposta de la citoquina TGF-[beta] la qual mostra una força dependència amb Snail1. Quan s’elimina Snail1 en ratolins adults provoca una clara disminució en el nombre de cèl•lules mare de la medul•la òssia. Aquestes cèl•lules en cultiu presenten una clara diferenciació prematura a osteoblasts i adipòcits. Pel contrari, tractaments amb TGF-[beta]1 aturen la diferenciació. El TGF-[beta]1 es incapaç de incrementar moltes dianes en cèl•lules mare del mesènquima aïllades del ratolí deficient per snail1, aquest efecte en part es degut a la repressió de PTEN i l’activació de AKT. L’anàlisi de sarcomes humans ens ha mostrat una alta expressió de Snail1, el qual també es troba associada amb una alta expressió de TGF-[beta] i baixa supervivència. Aquests resultats no només demostren una nova funció per Snail1 en resposta a TGF-[beta] i el manteniment de les MSC, sinó que també suggereix que Snail1 podria participar en la generació del sarcoma.

Glucocorticoids are widely used in clinical therapy. However, they cause adverse effects including insulin resistance and Type 2 diabetes, which are characterised by decreased glucose transport into the muscles and fat. How glucocorticoids inhibit glucose transport remains unclear. Insulin stimulates glucose uptake via the insulin receptor substrate (IRS)-1 / phosphoinositide-3-kinase (PI3K) / protein kinase B (AKT) pathway and promotes the redistribution of GLUT4 from intracellular storage compartments to the plasma membrane (PM). Insulin-stimulated phosphorylation of AKT substrate of 160 kDa (AS160), a Rab-GTPase activating protein is downstream of AKT and appears to be essential for exposure of GLUT4 at the PM and glucose uptake. This is mediated through the association of phosphorylated AS160 (at the key residue T642) with 14-3-3 in the cytosol. The mildly insulin-responsive GLUT1 mediates basal glucose uptake in adipocytes. It is also subject to regulated trafficking like GLUT4. This study aimed to determine the level at which glucocorticoids inhibit glucose uptake in adipocytes. Effects of the synthetic glucocorticoid dexamethasone (Dex) and the natural glucocorticoid cortisol, on GLUT1 and GLUT4 function were examined. Candidates for the glucocorticoid-mediated inhibition of GLUT1- and GLUT4-mediated glucose uptake were investigated. These were glycogen synthase kinase (GSK) 3β (an AKT substrate) for GLUT1-mediated glucose transport; and adaptor protein containing PH domain, PTB domain, and leucine zipper motif (APPL)-1 (an AKT-interacting protein) and AS160 for GLUT4-mediated glucose transport. Dex and cortisol significantly decreased basal glucose uptake by 50% (p<0.05) in SGBS and 3T3-L1 adipocytes. Similarly, insulin-stimulated glucose uptake was decreased by 50% (p<0.001 for SGBS; p<0.05 for 3T3-L1) and 30% (p<0.05 for both) at 1 nM and 100 nM insulin respectively. Similar results were observed with differentiated primary human preadipocytes and human adipose explants. Dex-mediated inhibition of basal glucose uptake was limited to insulin-sensitive cell types implying that glucocorticoids may regulate GLUTs at steps common to GLUT1 and GLUT4 trafficking. Dex-mediated reduction in glucose uptake correlated with the reduction in basal and insulin-stimulated expression of GLUT1 and GLUT4 to the PM without changes in total GLUT1/4 expression. Dex did not alter total expression or phosphorylation of proximal insulin-signalling molecules up to and including AKT but increased FOXO1 expression, and modified GSK3β-S9 phosphorylation. Dex did not alter total APPL1 expression or subcellular distribution. Dex significantly decreased 1nM-insulin stimulated AS160-T642 phosphorylation by 50% (p<0.05) in SGBS and 3T3-L1 adipocytes via the glucocorticoid repector (GR). This correlated with reduced AS160:14-3-3 interaction. Similar results were obtained for AS160-T642 basal phosphorylation. At 1nM insulin, AS160-T642 phosphorylation is maximal at sub-maximal glucose uptake, i.e. AS160 phosphorylation significantly contributes to glucose uptake. RU486 significantly prevented but did not fully abrogate the Dex-mediated reduction in glucose uptake suggesting additional Dex-induced defects. In conclusion, glucocorticoids inhibit glucose uptake at a level distal to AKT by GR-dependent mechanisms. A role for GSK3β or APPL1 in glucocorticoid-mediated inhibition of glucose uptake requires further investigations. FOXO1 represents a suitable candidate for mediating the Dex-induced defects. Of significance, perturbation in AS160-T642 phosphorylation contributes to Dex-mediated inhibition of glucose uptake. Thus, AS160 presents a novel therapeutic target in the improvement of glucocorticoid-mediated inhibition of glucose uptake.

Thesis (Ph.D. in Reproductive Sciences) -- University of Colorado Denver, 2008. / Typescript. Includes bibliographical references (leaves 150-183). Free to UCD Anschutz Medical Campus. Online version available via ProQuest Digital Dissertations;

Thesis (Ph.D. in Molecular Biology) -- University of Colorado at Denver and Health Sciences Center, 2006. / Typescript. Includes bibliographical references (leaves 194-209). Free to UCD Anschutz Medical Campus. Online version available via ProQuest Digital Dissertations;

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