Once seen as a disease of wealthy nations, type 2 diabetes mellitus is now showing unprecedented growth throughout the world, fueling increases in microvascular and macrovascular complications. A compelling and growing body of evidence suggests that glucose intolerance and insulin resistance, hallmarks of the diabetic patient, may be driven by chronic inflammation. In particular, a predominance of visceral fat has been associated with enhanced inflammatory cytokine secretion that may contribute to enhanced risk of diabetes and comorbid cardiovascular disease in these individuals. As a function of its potency and wide environmental and biological distribution, we hypothesized that bacterial lipopolysaccharide (LPS, also known as endotoxin) may promote adipose inflammation and concomitant metabolic dysfunction. Indeed, expression of the LPS receptor CD14 is enhanced on visceral adipocytes of ob/ob mice, paralleling enhanced IL-6 secretion ex vivo. Furthermore, rosiglitazonefed ob/obmice demonstrated a reduction in CD14 that coordinated with diminished IL-6 secretion, suggesting a basis for the touted anti-inflammatory effects of this commonly employed type 2 diabetes medication. Mice deficient in components of the LPS signaling cascade, namely CD14, TLR4, and MyD88, yielded adipocytes with markedly attenuated IL-6 secretion, corroborating the central importance of LPS in adipocyte inflammation and supporting the role of this signaling pathway in depot-specific inflammation. Despite the prominent role of LPS signaling in adipocyte inflammation, CD14-, TLR4-, and MyD88-deficient mice failed to show resistance to diet induced obesity. Surprisingly, cd14-/- and tlr4-/- mice had marked glucose intolerance without alteration in total weight or adipose accumulation. In contrast, myd88-/- mice revealed minor glucose intolerance only with high fat diet challenge at an advanced age despite being overtly obese. In cd14-/- and tlr4-/-, but not myd88-/-, mice, an exaggerated rebound to hypoglycemia was associated with enhanced norepinephrine secretion, which could be abrogated by the adrenergic β-blocker propranolol. The overlay of these mouse models reveals a divergence of phenotypes that demonstrate LPS signaling disruption may lead to glucose intolerance and insulin resistance in part due to enhanced sympathoadrenal tone, uncovering an essential role of innate immunity in physiological stress and its impact upon glucose homeostasis.

Breast cancer is the most common malignancy among women in the U.S. While many successful treatments exist for primary breast cancer, very few are available for patients with metastatic disease. The purpose of this study was to understand the role of Insulin Receptor Subtrate-2 (IRS-2) in breast cancer metastasis. IRS-2 belongs to the IRS family of cytoplasmic adaptor proteins that mediate signaling from cell surface receptors, many of which have been implicated in cancer. Although the IRS proteins are highly homologous in structure and have some complementary functions, growing evidence supports that the IRS proteins have unique roles in cancer. IRS-1 has been shown to promote tumor cell proliferation, while IRS-2 has been positively associated with cancer cell invasion, glycolysis and tumor metastasis. In the current work, we identified IRS-2 as a novel hypoxia-responsive gene in breast carcinoma cells. In contrast, IRS-1 expression does not increase in response to hypoxia, supporting the notion of their non-overlapping functions. Hypoxia promotes the adaptation and resistance of cancer cells to chemo- and radiation therapy, and also promotes tumor cell survival, invasion and metastasis by selecting for aggressive tumor cells that can survive under stressful low oxygen conditions. We have shown that IRS-2 upregulation in response to hypoxia promotes Akt signaling and tumor cell viability and invasion. We identified a cell context-dependent role for Hypoxia Inducible Factor (HIF) in the regulation of IRS-2 expression in hypoxia, with HIF-2 playing a more dominant role than HIF-1. We also demonstrate that binding of Snail, a regulator of the EMT, to the IRS-2 promoter keeps the chromatin in an open conformation that is permissive for HIF-dependent transcription of IRS-2 in hypoxia. IRS-2 is not upregulated by hypoxia in well-differentiated epithelial-like carcinoma cells that do not express Snail, implicating IRS-2 gene expression as part of the EMT programming. In summary, we have identified an endogenous mechanism by which cancer cells can shift the balance of IRS-1 and IRS-2 to favor IRS-2 expression and function, which promotes survival, invasion, and ultimately metastasis. Understanding the mechanism of IRS-2 regulation by hypoxia may reveal new therapeutic targets for metastatic breast cancer.

Diabetes mellitus comprises a cohort of genetic and metabolic diseases which are characterized by the hallmark symptom of hyperglycemia. Diabetic subtypes are based on their pathogenetic origins: the most prevalent subtypes are the autoimmune-mediated type 1 diabetes mellitus (T1DM) and the metabolic disease of type 2 diabetes mellitus (T2DM). Genetic factors are major contributory aspects to diabetes development, particularly in T2DM where there is close to 80% concordance rates between monozygotic twins. However, the functional state of the pancreatic β cell is of paramount importance to the development of diabetes. Perturbations that lead to β cell dysfunction impair insulin production and secretion and precede diabetes onset. The endoplasmic reticulum (ER) is a subcellular organelle network of tubes and cisternae with multifaceted roles in cellular metabolism. Alterations to ER function such as those begotten by the accumulation of misfolded and unfolded ER client proteins upset the ER homeostatic balance, leading to a condition termed ER stress. Subsequent sensing of ER stress by three ER transmembrane proteins, initiates an adaptive reaction to alleviate ER stress: this is known as the unfolded protein response (UPR). Divergent cascades of the UPR attempt to mitigate ER stress and restore ER homeostasis: Failing that, the UPR initiates pro-apoptotic pathways. The demand of insulin production on the β cell necessitates the presence of a highly functional ER. However, the consequence of dependence on the ER for insulin synthesis and secretion portends disaster for the functional state of the β cell. Disturbances to the ER that elicit ER stress and UPR activation causes β cell dysfunction and may lead to apoptosis. There are numerous well-characterized models of ER stress-mediated diabetes, including genetic mutations in UPR transducers and insulin. Recently, polymorphisms in Wolfram syndrome 1 (WFS1), an ER transmembrane protein involved in the UPR, were suggested to contribute to T2DM risk. In this thesis, one of the highlighted WFS1 polymorphism, H611R, was examined to identify its contribution to β cell function and viability, and hence, diabetes risk. It was revealed that augmentation of WFS1 expression increased insulin secretion and cellular content. In addition, WFS1 protected β cells against ER stress-mediated dysfunction, with a more pronounced effect in the WFS1-R611 protective allele. Subsequent gene expression analysis identified netrin-1 as a WFS1-induced survival factor. As a contributory factor to diabetes progression, ER stress and UPR are potential drug and biomarker targets. In this dissertation, a novel UPR-regulating microRNA (miRNA) family was uncovered in ER stressed, WFS1-deficient islets. These miRNAs, the miR-29 family, are induced in WFS1 -/- islets as a possible adaptive alteration to chronic ER stress conditions, and indirectly decreases the expression of UPR transducers, while directly targeting downstream ER stress-related pro-apoptotic factors. Collectively, this work extends the function of WFS1 as a protective factor in the pancreatic β cell through the induction of netrin-1 signaling. Additionally, it further strengthens the role of miRNA as regulatory members of the UPR which contribute to cell survival.

Indiana University-Purdue University Indianapolis (IUPUI). / Calcium/calmodulin-dependent protein kinase II (CaMKII) is a critical modulator of synaptic function, plasticity, and learning and memory. In neurons and astrocytes, CaMKII regulates cellular excitability, cytoskeletal structure, and cell metabolism. A rapid increase in CaMKII activity is observed within the first few minutes of ischemic stroke in vivo; this calcium-dependent process is also observed following glutamate stimulation in vitro. Activation of CaMKII during pathological conditions is immediately followed by inactivation and aggregation of the kinase. The extent of CaMKII inactivation is directly correlated with the extent of neuronal damage. The studies presented here show that these fluctuations in CaMKII activity are not correlated with neuronal death; rather, they play a causal role in neuronal death. Pharmacological inhibition of CaMKII in the time immediately surrounding glutamate insult protects cultured cortical neurons from excitotoxicity. Interestingly, pharmacological inhibition of CaMKII during excitotoxic insult also prevents the aggregation and prolonged inactivation of the kinase, suggesting that CaMKII activity during excitotoxic glutamate signaling is detrimental to neuronal viability because it leads to a prolonged loss of CaMKII activity, culminating in neuronal death. In support of this, CaMKII inhibition in the absence of excitotoxic insult induces cortical neuron apoptosis by dysregulating intracellular calcium homeostasis and increasing excitatory glutamate signaling. Blockade of the NMDA-receptors and enzymatic degradation of the extracellular glutamate signal affords neuroprotection from CaMKII inhibition-induced toxicity. Co-cultures of neurons and glutamate-buffering astrocytes also exhibit this slow-induced excitotoxicity, as CaMKII inhibitors reduce glutamate uptake within the astrocytes. CaMKII inhibition also dysregulates calcium homeostasis in astrocytes and leads to increased ATP release, which was neurotoxic when applied to naïve cortical neurons. Together, these findings indicate that during aberrant calcium signaling, the activation of CaMKII is toxic because it supports aggregation and prolonged inactivation of the kinase. Without CaMKII activity, neurons and astrocytes release stores of transmitters that further exacerbate neuronal toxicity.

Le système rénine-angiotensine-aldostérone est impliqué dans la physiopathologie de l’insuffisance cardiaque (IC). L’inhibition de l’aldostérone par les antagonistes du récepteur aux minéralocorticoïdes (ARM), la spironolactone et l’éplérénone, est associée à une réduction de morbidité et mortalité. Or, la spironolactone est un antagoniste non sélectif, avec des effets hors-cibles sur d’autres récepteurs stéroïdiens. Des données suggèrent qu’elle pourrait avoir un effet défavorable sur l’homéostasie du glucose, avec une augmentation en hémoglobine glyquée (HbA1c), un marqueur de contrôle du glucose à long terme. Au contraire, l’éplérénone semble exercer un effet neutre. Les objectifs de cette thèse de doctorat sont les suivants : 1) Assembler toutes les connaissances dans la littérature au sujet de l’effet glycémique des ARM; 2) Évaluer le risque de développement de diabète avec la spironolactone chez les patients IC; 3) Analyser si la spironolactone peut moduler l’effet glycémique d’autres médicaments utilisés en IC; 4) Comparer la spironolactone à l’éplérénone sur des marqueurs de glucose chez les patients IC avec dérèglements glycémiques. Quatre projets ont été effectués afin de répondre à ces questions. Premièrement, une revue systématique a permis d’identifier toutes les études publiées contenant de l’information sur l’effet glycémique des ARM. Les résultats étaient hétérogènes, mais ont suggéré que l’effet est dépendant de la pathologie et serait potentiellement néfaste dans les maladies à haut risque d’évènements cardiovasculaires. Une méta-analyse d’études en diabète indique que l’effet à long terme serait non significatif. Le deuxième projet utilise une cohorte de patients IC de bases de données administratives entre 1995 et 2009 (suivi jusqu’en 2010). Nous n’avons pas détecté d’association significative entre l’utilisation de la spironolactone et le risque de diabète. Par contre, l’étude a démontré qu’un âge plus jeune, la digoxine, et les corticostéroïdes augmentent le risque de diabète. Le troisième projet est une sous-étude d’une étude clinique CANDIID-II (Effect of ACE inhibitor alone versus ACE inhibitor plus high dose candesartan on BNP, immune markers, inflammatory status, and urinary kinins in patients with symptomatic left ventricular systolic dysfunction) chez des patients IC traités avec un inhibiteur de l’enzyme de conversion à l’angiotensine et le candésartan, antagoniste du récepteur à l’angiotensine II. Ces classes pharmacologiques ont des effets bénéfiques sur la glycémie. En comparant les patients traités aussi avec la spironolactone versus les patients sans ARM, nous n’avons pas trouvé que la spironolactone module l’effet bénéfique du candésartan sur le métabolisme du glucose. Le dernier projet consiste d’une étude prospective, multicentrique, randomisée, contrôlée à double-insu : SNOW (A comparison of the effects of selective and non selective mineralocorticoid antagonism on glucose homeostasis and lipid profile of heart failure patients with glucose intolerance or type 2 diabetes). Elle compare, pendant 16 semaines, la spironolactone à l’éplérénone sur des marqueurs glycémiques, notamment, l’HbA1c, chez 62 patients IC avec diabète de type II ou intolérance au glucose. Aucune différence significative n’a été observée entre les groupes. En résumé, les résultats de cette thèse indiquent que les ARM ne présentent pas de risque de détérioration du contrôle du glucose sur une durée modérée à longue en IC. / The renin-angiotensin-aldosterone system is involved in the pathophysiology of heart failure (HF). The inhibition of aldosterone by mineralocorticoid receptor antagonists (MRAs), spironolactone and eplerenone, is associated with a reduction in morbidity and mortality. However, spironolactone is a non selective antagonist, with off-target effects on other steroid receptors. There is some evidence suggesting that it may have an unfavorable effect on glucose homeostasis, with an increase in glycated hemoglobin (HbA1c), a marker of long-term glucose control. On the contrary, eplerenone seems to exert a neutral effect. The objectives of this doctoral thesis were the following: 1) Compile all current knowledge in the literature on the subject of MRAs’ glycemic effects; 2) Evaluate the risk of developing diabetes with spironolactone in HF patients; 3) Analyze if spironolactone may modulate the glycemic effects of other medications used in HF; 4) Compare spironolactone to eplerenone on markers of glucose control in HF patients with glycemic disorders. Four projects were conducted in order to meet these objectives. Firstly, a systematic review allowed us to identify all published studies containing information on MRAs’ glycemic effects. The literature search yielded heterogenous results; however, it suggested that the effect was disease-specific and would be potentially harmful in diseases with a high risk of cardiovascular events. A meta-analysis of studies in diabetes insinuated that the effect is non significant on a long-term basis. The second project uses a cohort of HF patients from administrative databases between 1995 and 2009 (follow-up till 2010). We did not detect a significant association between the use of spironolactone and the risk of diabetes. On the other hand, the study demonstrated that younger age, digoxin, and corticosteroids increase the risk of diabetes. The third project is a substudy of a clinical trial CANDIID-II (Effect of ACE inhibitor alone versus ACE inhibitor plus high dose candesartan on BNP, immune markers, inflammatory status, and urinary kinins in patients with symptomatic left ventricular systolic dysfunction) among HF patients treated with an angiotensin converting enzyme inhibitor and candesartan, an angiotensin II receptor blocker. These pharmacological classes have beneficial effects on glycemia. By comparing patients also treated with spironolactone versus patients without an MRA, we did not find that spironolactone alters the effect of candesartan on glucose metabolism. The last project consisted of a prospective, multicenter, randomized, controlled, double-blind trial: SNOW (A comparison of the effects of selective and non selective mineralocorticoid antagonism on glucose homeostasis and lipid profile of heart failure patients with glucose intolerance or type 2 diabetes). It compares, for 16 weeks, spironolactone to eplerenone on glycemic markers, notably, HbA1c, among 62 HF patients with type II diabetes or glucose intolerance. There was no significant difference between groups. In summary, the research results from this thesis reveal that, in HF, MRAs do not present additional risks of deterioration in glucose control over a moderate to long period.

No / The alpha-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate receptor (AMPAR) subunit GluR2, which regulates excitotoxicity and the inflammatory cytokine tumour necrosis factor alpha (TNFalpha) have both been implicated in motor neurone vulnerability in amyotrophic lateral sclerosis/motor neurone disease. TNFalpha has been reported to increase cell surface expression of AMPAR subunits to increase synaptic strength and enhance excitotoxicity, but whether this mechanism occurs in motor neurones is unknown. We used primary cultures of mouse motor neurones and cortical neurones to examine the interaction between TNFalpha receptor activation, GluR2 availability, AMPAR-mediated calcium entry and susceptibility to excitotoxicity. Short exposure to a physiologically relevant concentration of TNFalpha (10 ng/mL, 15 min) caused a marked redistribution of both GluR1 and GluR2 to the cell surface as determined by cell surface biotinylation and immunofluorescence. Using fura-2-acetoxymethyl ester microfluorimetry, we showed that exposure to TNFalpha caused a rapid reduction in the peak amplitude of AMPA-mediated calcium entry in a PI3-kinase and p38 kinase-dependent manner, consistent with increased insertion of GluR2-containing AMPAR into the plasma membrane. This resulted in a protection of motor neurones against kainate-induced cell death. Our data therefore, suggest that TNFalpha acts primarily as a physiological regulator of synaptic activity in motor neurones rather than a pathological drive in amyotrophic lateral sclerosis.

No / Bone morphogenetic proteins (BMPs) are a large family of multi-functional secreted signalling molecules. Previously BMP2/4 were shown to inhibit skin pigmentation by downregulating tyrosinase expression and activity in epidermal melanocytes. However, a possible role for other BMP family members and their antagonists in melanogenesis has not yet been explored. In this study we show that BMP4 and BMP6, from two different BMP subclasses, and their antagonists noggin and sclerostin were variably expressed in melanocytes and keratinocytes in human skin. We further examined their involvement in melanogenesis and melanin transfer using fully matched primary cultures of adult human melanocytes and keratinocytes. BMP6 markedly stimulated melanogenesis by upregulating tyrosinase expression and activity, and also stimulated the formation of filopodia and Myosin-X expression in melanocytes, which was associated with increased melanosome transfer from melanocytes to keratinocytes. BMP4, by contrast, inhibited melanin synthesis and transfer to below baseline levels. These findings were confirmed using siRNA knockdown of BMP receptors BMPR1A/1B or of Myosin-X, as well as by incubating cells with the antagonists noggin and sclerostin. While BMP6 was found to use the p38MAPK pathway to regulate melanogenesis in human melanocytes independently of the Smad pathway, p38MAPK, PI3-K and Smad pathways were all involved in BMP6-mediated melanin transfer. This suggests that pigment formation may be regulated independently of pigment transfer. These data reveal a complex involvement of regulation of different members of the BMP family, their antagonists and inhibitory Smads, in melanocytes behaviour.

Der wichtigste exzitatorische Neurotransmitter Glutamat spielt eine grosse Rolle in der Gehirnentwicklung, wie neuronale Migration und Synaptogenese. Ob glutamaterge Stimulation für das Überleben entwickelnder Neuronen notwendig ist, war bislang jedoch unbekannt. Um zu untersuchen, ob eine Hemmung von Glutamatrezeptoren im unreifen Gehirn zu Neurodegeneration führt, wurden Ratten im Alter von 1 bis 31 Tagen für 24 Stunden mit dem N-Methyl-D-Aspartat-(NMDA) Glutamatrezeptorantagonisten Dizocilpin (MK801) behandelt. Die Dichte neuronaler Degeneration wurde mikroskopisch in Kupfer-Silber- und TUNEL- gefärbten Hirnschnittpräparaten ermittelt und Unterschiede mittels ANOVA analysiert (Signifikanzniveau p / The predominant excitatory neurotransmitter glutamate plays a major role in certain aspects of neural development. However, whether developing neurons depend on glutamate for survival remains unknown. To investigate if deprivation of glutamate stimulation in the immature mammalian brain causes neuronal cell death (apoptosis), rat pups aged 0 to 30 days were treated for 24 hours with dizocilpine maleate (MK801), an N-methyl-D-aspartate-(NMDA) glutamate receptor antagonist. Density of neural degeneration was evaluated by a stereological dissector method in cupric-silver and TUNEL-stained brain slices. Groups were compared by ANOVA and significance considered at p

Cardiovascular disease (CVD) is the leading cause of death worldwide and is partially attributed to perturbations in lipid metabolism. Xenobiotics, such as pharmaceutical drugs and environmental chemicals, have been associated with increased risk of CVD in multiple large-scale human population studies, but the underlying mechanisms remain poorly defined. We and others have identified several xenobiotics as potent agonists for the pregnane X receptor (PXR), a nuclear receptor that can be activated by numerous drugs as well as environmental and dietary chemicals. However, the role of PXR in mediating the pathophysiological effects of xenobiotic exposure in humans and animals remains elusive. The work herein identified several widely used pharmaceutical agents and endocrine disrupting chemicals as PXR-selective agonists such as drugs involved in HIV therapy and phthalates/phthalate substitutes, respectively. We investigated the role of amprenavir, an HIV protease inhibitor, and tributyl citrate, a phthalate substitute, on PXR-dependent alterations in lipoprotein metabolism. Acute exposure with either xenobiotic in mice elicited increases in the proatherogenic LDL-cholesterol levels in a PXR-dependent manner. PXR activation significantly induced expression of genes involved in intestinal lipid metabolism. Further, we went on to identify the intestinal cholesterol transporter, Niemann-Pick C1-Like 1 (NPC1L1), as a direct PXR-target gene. PXR activation also stimulated cholesterol uptake in both murine and human intestinal cells. Moreover, we provide evidence that the microsomal triglyceride transfer protein (MTP) may be a direct PXR-target gene. Taken together, these findings provide critical mechanistic insight into the role of xenobiotic-mediated PXR activation on lipid homeostasis and demonstrate a potential role of PXR in mediating adverse effects of xenobiotics on CVD risk in humans. In addition to PXR signaling, we investigated the role of IκB kinase β (IKKβ), a central coordinator of inflammation, in adipocyte progenitor cells. Targeting IKKβ in adipose progenitor cells resulted in decreased high fat diet (HFD)-elicited adipogenesis, while protecting mice from inflammation and associated insulin resistance. Consistently, we discovered that IKKβ inhibition by antisense oligonucleotides ablated HFD-induced adiposity, while protecting mice against associated metabolic disorders. In conclusion, targeting IKKβ with antisense therapy may present as a novel therapeutic approach to combat obesity and metabolic dysfunctions.

Objetivo: Comparar o efeito da terapia da fotocoagulação panretiniana (PFC) associada à injeção intravítrea de Ranibizumabe (RBZ) versus terapia isolada com PFC em pacientes com retinopatia diabética proliferativa (RDP) precoce, virgens de tratamento, com ou sem edema macular diabético (DME) durante 6 meses de acompanhamento. Projeto: Estudo prospectivo intervencionista, randomizado e controlado. Métodos: Sessenta olhos de 30 pacientes com RDP bilateral precoce foram randomizados para o grupo de estudo (GE) que foram tratados com PFC associado a duas injeções de RBZ intravítreo (0.5mg/0.05ml) ou para o grupo controle (GC) tratados apenas com PFC. Mudanças na acuidade visual (AV) corrigida, na sensibilidade ao contraste (SC) e na espessura foveal (EF) foram comparados no início, e nos 1, 3 e 6 meses após o tratamento. Resultados: No GE, a diferença na média da AV do baseline para o mês 6 teve um aumento significativo de + 3,4 letras (p = 0,006) e uma diminuição significativa na EF de - 47.6um (p < 0,001). No GC, a diferença na média da AV teve uma diminuição de - 3,4 letras (p = 0,04) e uma mudança na EF de -3.8 um (p = 0,96). Com relação ao teste de SC dentre os 28 olhos do GE, houve uma melhora no mês 6 em relação ao baseline nos ciclos: 1,5 (p < 0.001) e 3,0 ciclo (p=0.023). Dentre os 30 olhos do GC, não houve uma diferença estatística nos momentos estudados. Conclusão: A injeção intravítrea de RBZ associado com PFC pode ser um tratamento eficaz em olhos de pacientes com RDP precoce e EMD / Purpose: To compare the efficacy of therapy with panretinal photocoagulation (PRP) and intravitreal ranibizumab (RBZ) injection versus PRP alone in patients with treatment-naive bilateral non-high risk proliferative diabetic retinopathy (PDR) with and without diabetic macular edema (DME) with a 6-month follow-up. Design: Prospective, interventional, randomized controlled trial. Methods: Sixty eyes of 30 patients with bilateral non-high risk PDR were randomized either to the study group (SG) receiving PRP plus two intravitreal ranibizumab injections (0.5mg/0.05ml), the first one week before and the second four weeks after the PRP or to the control group (CG) receiving PRP alone. Mean change in best-corrected visual acuity (BCVA), contrast sensitivity (CS) and central macular thickness (CMT) were compared at baseline and 1, 3 and 6 months after treatment. Results: Changes from baseline to 6 months showed in the SG an increased in the BCVA by + 3.4 letters (p= 0.006) with a decrease in CMT by - 47.6um (p < 0.001). In the CG, a decrease by - 3.4 letters (p = 0.04) and an decrease by -3.8um (p= 0.96). Regarding the CS in the SG, there was an improvement compared to baseline for the sixth month in the 1.5 (p < 0.001) and 3.0 cycles (p = 0.023). The CG did not show significant results from baseline to month 6. Conclusion: Intravitreal RBZ associated with PRP can be an effective treatment in eyes with non-high risk PDR and DME