Background & Aims: The 'high-density lipoprotein (HDL) hypothesis', that therapeutic interventions directed at raising HDL cholesterol might translate into improved cardiovascular outcomes, has been confounded by recent reports from genetic and pharmacological studies. HDL functionality may be more important than cholesterol cargo. HDL cholesterol levels are normal or even high in Type 1 Diabetes (T1DM) but do not seem to protect against atherosclerosis as might be expected; this thesis aims to offer new insight into HDL functionality through examination of these patients. This thesis also aims to improve understanding of the qualitative changes in lipoproteins associated with diabetes and increased cardiovascular morbidity, with emphasis on atherogenic modifications of apolipoprotein B and sphingolipids, and consideration of the relationship between these changes, novel and established biomarkers, and macrovascular and microvascular diabetic complications. Materials & Methods: Patients with Type 1 (n = 91) and Type 2 (n = 40) Diabetes Mellitus and healthy volunteers (n = 104) attended for fasting blood tests, urinalysis, and examination including cardiac computed tomography, carotid doppler studies and assessments of nerve function. In vitro studies of lipoprotein modification used pooled human plasma. Results: Lipoprotein glycation represents an atherogenic modification. In vitro glycation occurs more readily in the presence of physiological concentrations of copper. HDL and copper-selective chelation with triethylenetetramine prevents glycation. Glycated apolipoprotein B, oxidized LDL and small-dense LDL levels were significantly higher in T1DM; HDL cholesterol levels were also significantly higher, but with altered apolipoprotein distribution, and significantly lower cholesterol efflux capacity and PON1 activity than in healthy controls. Significant changes were also observed in cystatin C, advanced glycation end-products, leucine-rich alpha-2-glycoprotein, lipoprotein-associated phospholipase A2, a variety of inflammatory markers, and sphingolipid and ceramide profiles. Discussion: Cardiovascular disease is the leading cause of death and disability in diabetes. Patients with diabetes show qualitative and kinetic lipoprotein abnormalities, and any cardiovascular benefit associated with intensive glucose lowering may be related to effects on lipoprotein metabolism rather than directly through altered glycaemia. The apparently relatively undisturbed lipid profile observed in many patients with diabetes hides major atherogenic changes and altered HDL functionality, which may be at least partially responsible for the persistent increased risk of cardiovascular disease in patients with diabetes. HDL-based therapy remains a largely unfulfilled promise, but there may be a role for copper-selective chelation and more aggressive low-density lipoprotein lowering in the reduction of diabetic complications.

Orientador: Fernando Antonio Santos Coelho / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Química / Made available in DSpace on 2018-08-21T16:21:23Z (GMT). No. of bitstreams: 1 Yamakawa_NathaliaChristinaGoncalves_M.pdf: 16524766 bytes, checksum: 72cb2d423e322cd66bf985a8a162ae5a (MD5) Previous issue date: 2012 / Resumo: A epilepsia é uma das principais doenças do sistema nervoso central, caracterizada por uma alteração na atividade elétrica do cérebro, que leva a perdas de memória e convulsões. Essa doença afeta cerca de 50 milhões de pessoas no mundo, que são discriminadas e isoladas da sociedade. Existem vários medicamentos que podem ser utilizados, no entanto, muitas vezes é necessário a administração de associações desses que apresentam severos efeitos colaterais. Esse quadro justifica a necessidade da busca por substâncias mais eficientes para o tratamento dessa doença. Em 2008, Ahmed e cols. isolaram da esponja marinha Negombata corticata duas ceramidas que apresentaram importante atividade anti-convulsiva. Assim, este trabalho teve por objetivo estabelecer uma estratégia sintética para a preparação do fragmento polar de tais ceramidas. A rota foi baseada em um aduto de Morita-Baylis-Hillman (MBH), obtido com elevada diastereosseletividade a partir de uma reação entre o aldeído de Garner e o acrilato de etila. Uma reação de ozonólise na dupla ligação do aduto de MBH fornece um a-cetoéster, cuja carbonila cetônica e reduzida conduzindo a um único produto. A proteção das hidroxilas, permitiu a confirmação da estereoquímica relativa através de análise por difração de raios-X, que também evidenciou a ocorrência de racemização na reação de MBH. O diol também foi utilizado na preparação de um aza-açúcar, potencial inibidor de glicosidase, sendo esta síntese em apenas 4 etapas com 32% de rendimento global. A fração apolar da ceramida foi sintetizada a partir de uma reação de Grignard, e a junção dos fragmentos pode ser realizada utilizando-se uma reação de Wittig. Desta maneira, foi descrita uma nova estratégia sintética que pode ser aplicada na preparação de diversos análogos das ceramidas, desenvolvendo um antiepiléptico mais potente / Abstract: Epilepsy is a chronic brain disorder that affects around 50 million people all over the world. It is characterized by recurrent seizures - which are physical reactions to sudden excessive electrical discharges in a group of brain cells. The discrimination and social stigma that surround epilepsy worldwide are often more difficult to overcome than the seizures themselves. Because of this fact and the economical impacts of the disease, the research for new biologically active compounds is still necessary. In 2008, Ahmed et al. isolated from the Red Sea sponge Negombata corticata two ceramides, which exhibit in vivo anticonvulsant activity. This work is focused on establishing of a synthetic sequence to prepare the polar fragment present in both ceramides. The strategy was based on a Morita-Baylis-Hillman reaction between a Garner¿s aldehyde and ethyl acrylate that provided a functionalized intermediate in good diastereoselectivity. The major diastereoisomer was employed as substrate in an ozonolysis reaction, followed by a stereoselective reduction that afforded 1,2-diol as a single isomer. The acetonide derived from this 1,2-diol allowed us to determine through X-Ray diffraction analysis the relative stereochemistry of this compound as being 1,2- anti. To finish the synthesis of the polar fragment, the ester group present in the acetonide was reduced to the corresponding aldehyde. The diol also was applied in a high diastereoselective preparation of an azasugar in 4 steps and 32% yield overall. In this work we also describe the synthesis of a carbon chain of the ceramide, our route includes an approach to the apolar fragment obtained by a Grignard reaction; then a Wittig reaction can couple both fragments toward the finalization of the sphingosine¿s synthesis. Our synthetic route can also be used in the preparation of several analogues of the antiepiletic ceramides / Mestrado / Quimica Organica / Mestre em Química

Charles University, Faculty of pharmacy in Hradec Králové Department of Organic and Bioorganic Chemistry Candidate: Linda Svatošová Supervisor: PharmDr. Lukáš Opálka, Ph.D. Title of diploma thesis: The study of model lipid membranes containing omega- hydroxylated ceramides Acylceramides (EO-Cer) belong to a class of ceramides (Cer) with an ultralong acyl chain whose ω-hydroxyl group is esterified with linoleic acid. The importance of EO-Cer lies in the formation of the long periodicity phase (LPP) and the corneocyte lipid envelope (CLE), which are indispensable components for the skin functioning as a barrier. Disorders in EO-Cer biosynthesis are associated with insufficient production of CLE and LPP leading to many skin diseases, including some types of ichthyosis. One of the enzymes that is deficient in such ichthyoses is PNPLA1. Insufficient function of this enzyme disables ω-esterification with linoleic acid, and thus the formation of EO-Cer. On the contrary, their precursors, i.e. ω-hydroxylated ceramides (O-Cer), are cumulated. The aim of this thesis was to prepare model membranes containing O-Cer and to study the effects of O-Cer on the lipid organization and barrier properties of model membranes. Within this thesis, two types of membranes were prepared - the first type were membranes...

Indiana University-Purdue University Indianapolis (IUPUI) / The complex pathogenesis of emphysema involves disappearance of alveolar structures, in part attributed to alveolar cell apoptosis. The mechanism by which cigarette smoke (CS) induces alveolar cell apoptosis is not known. We hypothesized that ceramides are induced by CS via specific enzymatic pathways that can be manipulated to reduce lung cell apoptosis. CS increased ceramides in the whole lung and in cultured primary structural lung cells. Exposure to CS activated within minutes the acid sphingomyelinase, and within weeks the de novo- ceramide synthesis pathways. Pharmacological inhibition of acid sphingomyelinase significantly attenuated CS-induced apoptosis. To understand the mechanisms by which ceramides induce apoptosis, we investigated the cell types affected and the involvement of RTP801, a CS-induced pro-apoptotic and pro-inflammatory protein. Direct lung augmentation of ceramide caused apoptosis of both endothelial and epithelial type II cells. Ceramide upregulated RTP801 and the transgenic loss of RTP801 inhibited only epithelial, but not endothelial cell apoptosis induced by ceramide. In conclusion, CS induces acid sphingomyelinase-mediated ceramide upregulation and apoptosis in a cell-specific manner, which in epithelial cells involves induction of stress response proteins that may further amplify lung injury. Molecular targeting of amplification pathways may provide therapeutic opportunities to halt emphysema progression.

Esponjas do gênero Dysidea (Ordem: Dictyoceratida) caracterizam-se por apresentarem grande diversidade de metabólitos secundários, muitos dos quais apresentam potentes atividades biológicas. Este trabalho descreve o estudo de duas amostras de esponjas da espécie Dysidea robusta, DR1 e DR2, coletadas no litoral da Bahia em 1999. Tal estudo consistiu no fracionamento das amostras, nas análises de seus extratos brutos por LC-MS e técnicas de RMN- mono e bidimensionais. Dentre os extratos de DR1, a fração DR1-EP-5A obtida do extrato éter de petróleo apresentou uma mistura de três ceramidas saturadas (22, 23 e 24). Já da amostra DR2, as frações do extrato aquoso DR2-AQ-6B e DR2-AQ-6D mostraram ser constituídas por derivados do ácido pirodisinóico (18, 19, 20 e 21). Com exceção do ácido pirodisinóico (18), os demais compostos isolados ainda não foram relatados na literatura. / Sponges of the genus Dysidea (Order: Dyctioceratida) are characterized as sources of several biologically active secondary metabolites. This work describes the study of two samples of D.robusta, DR1 and DR2, both collected at the Bahia state coastline, in 1999. The investigation aimed the crude extract fractionation and analysis by LC-MS and by 1D and 2D NMR techniques. Among the extracts DR1, the fraction DR1-EP-5A obtained from the petroleum ether extract showed a mixture of three saturated ceramides, represented by 22, 23 and 24. From the DR2 sample, the fractions obtained from the aqueous extract DR2-AQ-6B and -6D presented pirodisinoic acid derivates 18, 19, 20 and 21. Except for pyrodisinoic acid (18), all other isolated compounds haven´t been reported in the literature yet.

Besides playing its structural function in cellular membranes, ceramide has been recognized as a bioactive signalling molecule playing roles in the regulation of cell growth, differentiation, senescence and programmed cell death. Apoptosis can be induced in cancer cells by elevation of endogenous ceramide levels in response to a variety of apoptotic stimuli such as cytokines (TNF, IL-1), death receptor ligands (Fas ligand), heat stress, oxidative stress, chemotherapeutic agents, and ionizing or ultraviolet radiation. It was shown that use of exogenous cell-permeable short-chain ceramide can also promote apoptotic pathways in cancer cells. Several studies have attempted to further define the specific role of ceramide in cell death. However, the mechanisms by which ceramide mediates antiproliferative pathways or inhibits prosurvival effects are not yet well-defined. So, we investigated the signalling pathways triggered by exogenously-supplied natural long chain ceramide, especially C16-ceramide, to better understand how this messenger induces its biological effects in cancer cells. We first showed that C16-ceramide induced a decrease in viability of adenocarcinoma cells (HCT116), partly due to apoptosis. Using two-dimensional differential in-gel electrophoresis (2D-DIGE) proteomic approach, we identified new proteins involved notably in cell proliferation, apoptosis, protein transport and transcriptional regulation in response to exogenous C16-ceramide. Among them, the death promoting factor Btf (Bcl-2-associated transcription factor) was found to be involved in the ceramide-dependent pro-apoptotic signalling pathway. Indeed, Btf-depleted colon cancer cells were found to be more resistant to death triggered by C16-ceramide. Transfection of GFP-Btf expression plasmid up-regulated p53 and BAX protein levels whereas pBcl-2 and Mdm2 expression were down-regulated. Furthermore, we identified a new signalling pathway specifically induced by C16-ceramide, depending on Btf and leading to down-regulation of the Mdm2 protein expression and MDM2 promoter activity. Thus, we provided new information on molecular mechanisms involved in the ceramide-mediated cell death. Then, we investigated the regulation of Emerin expression and its post-translational modifications induced by ceramide. We found that cAMP-dependent protein kinase A (PKA) could be involved in the C16-ceramide induced-Emerin phosphorylation. However, we did not demonstrate the interaction between Btf and phosphorylated-Emerin upon ceramide treatment. Nevertheless, we showed that one of the pathway induced by ceramide implies Emerin and leads to down-regulation of the MDM2 promoter activity. We also hypothesized that GCL (germ-cell-less) could be an intermediate in the Emerin-Mdm2 pathway triggered by C16-ceramide. Furthermore, we showed that Emerin-depleted cells were not more sensitive to apoptosis induced by C16-ceramide. These results should allow us to further explore the potential functions of Emerin in a ceramide-dependent pathway.

Thesis (Ph.D)--Biology, Georgia Institute of Technology, 2009. / Committee Chair: Alfred H. Merrill, Jr; Committee Member: John Cairney; Committee Member: M. Cameron Sullards; Committee Member: Marion B. Sewer; Committee Member: Yuhong Fan. Part of the SMARTech Electronic Thesis and Dissertation Collection.

Insulin resistance is commonly developed in obesity and is a trait of the beginning stage of type 2 diabetes mellitus (DM). It is highly likely that the high plasma fatty acid levels provoke the condition in the obese and insulin resistant state of type 2 DM. Hence, the purpose of this study was to determine if a high concentration of palmitic acid causes insulin resistance and how ceramide content is regulated under the various conditions in the isolated rat soleus muscle. A submaximal insulin stimulus (100 ,aU/ml) increased 3-O-methylglucose transport by -2.7 fold over basal conditions in the soleus (1.90 ± 0.23 µmol•ml"1•hr-1 vs. 5.06 ± 0.38 µmol•ml-1•hr-1, respectively) (P < 0.05). Five hours of palmitic acid preincubation induced a significant decrease in insulin-stimulated glucose transport (3.49 ± 0.11 µmol•ml-1•hr-1) by -31 % (P < 0.05) compared with the control. By contrast, the addition of L-cycloserine, a serine palmitoyltransferase inhibitor, attenuated the palmitic acid response by -20% (4.19 ± 0.27 µmol•ml-1•hr-1) (P < 0.05). A 5 hr preincubation with 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR), an adenosine analogue that increase AMP activated protein kinase, increased glucose transport (3.29 ± 0.1 µmol•ml-1•hr-1) (P < 0.05) compared with the control group. Moreover, regardless if palmitic acid or L-cycloserine were present, insulin-stimulated glucose uptake was normalized (5.30 ± 0.38 µmol•ml-1•hr-1 and 5.56 ± 0.16 µmol•ml"1•hr-1, respectively) after 5 hr AICAR preincubation. We next measured the ceramide content to investigate whether the reduced glucose uptake results from ceramide accumulation in the soleus. The total ceramide mass in the soleus was increased by -35% in palmitic acid-treated group compared with the control group (122.02 ± 2.07 pmol•mg-1 vs. 90.79 + 1.24 pmol•mg 1, respectively) (P<0.05). Both L-cycloserine and AICAR decreased palmitateinduced ceramide synthesis by -20% and -14%, respectively (97.15 + 2.5 pmol•mg-1 and 105.79 ± 1.94 pmol•mg-1, respectively) (P<0.05) compared with the palmitic acid-treated group. We also measured serine palmitoyltransferase (SPT) to determine if AICAR regulates ceramide synthesis by inhibiting SPT. Total SPT protein level increased by -27% (P<0.05) and SPT activity increased by -44% (P<0.05) compared with the control group. By contrast, after muscles were incubated with L-cycloserine for 5 hr, both SPT protein level and enzyme activity were decreased by -17% and -23%, respectively (P<0.05). In adition, 5 hr AICAR treatment blunted palimitic acid-induced SPT protein level and enzyme activity by 11% (P<0.05) and 20% (P<0.05), respectively, compared with the palmitic acid-treated group. In conclusion, these data suggest that short term exposure (5 hr) to high fatty acid levels appears to cause insulin resistance by increasing ceramide accumulation and that AMPK expression (AICAR treatment) can attenuate the problem by regulating SPT levels. / School of Physical Education, Sport, and Exercise Science

Esponjas do gênero Dysidea (Ordem: Dictyoceratida) caracterizam-se por apresentarem grande diversidade de metabólitos secundários, muitos dos quais apresentam potentes atividades biológicas. Este trabalho descreve o estudo de duas amostras de esponjas da espécie Dysidea robusta, DR1 e DR2, coletadas no litoral da Bahia em 1999. Tal estudo consistiu no fracionamento das amostras, nas análises de seus extratos brutos por LC-MS e técnicas de RMN- mono e bidimensionais. Dentre os extratos de DR1, a fração DR1-EP-5A obtida do extrato éter de petróleo apresentou uma mistura de três ceramidas saturadas (22, 23 e 24). Já da amostra DR2, as frações do extrato aquoso DR2-AQ-6B e DR2-AQ-6D mostraram ser constituídas por derivados do ácido pirodisinóico (18, 19, 20 e 21). Com exceção do ácido pirodisinóico (18), os demais compostos isolados ainda não foram relatados na literatura. / Sponges of the genus Dysidea (Order: Dyctioceratida) are characterized as sources of several biologically active secondary metabolites. This work describes the study of two samples of D.robusta, DR1 and DR2, both collected at the Bahia state coastline, in 1999. The investigation aimed the crude extract fractionation and analysis by LC-MS and by 1D and 2D NMR techniques. Among the extracts DR1, the fraction DR1-EP-5A obtained from the petroleum ether extract showed a mixture of three saturated ceramides, represented by 22, 23 and 24. From the DR2 sample, the fractions obtained from the aqueous extract DR2-AQ-6B and -6D presented pirodisinoic acid derivates 18, 19, 20 and 21. Except for pyrodisinoic acid (18), all other isolated compounds haven´t been reported in the literature yet.

L'obésité et le diabète de type 2 sont associés à la sédentarité et à une alimentation riche en graisses. En effet, les acides gras saturés s'accumulent dans les tissus non adipeux, comme les muscles squelettiques pour générer des lipides appelés céramides (CER). Mon projet de thèse s'est articulé en deux parties dont l'objectif est d'empêcher les CER d'agir. Nous avons montré que, selon la structure de la membrane plasmique, les CER altèrent la voie de signalisation insulinique en ciblant la PKB, protéine clef de la voie insulinique, via la voie PKC? dans les myotubes L6 et la voie PP2A dans les myotubes C2C12. Nous avons aussi mis en évidence que les CER altèrent la sensibilité à l'insuline via la voie PKC? dans les cellules musculaires humaines. Une fois les CER produits au niveau du réticulum endoplasmique (RE), ils sont transportés au Golgi par un transporteur CERT pour y être métabolisés en sphingomyéline (SM) et des études ont montré que la transformation des CER en SM pouvait être une étape cruciale pour empêcher les CER d'agir. Dans plusieurs modèles d'insulino-résistance musculaire, l'expression de CERT est diminuée et nous avons démontré l'importance du transport des céramides du RE vers le Golgi en inhibant artificiellement l'activité ou l'expression de CERT. A l'opposé, la surexpression de CERT améliore la sensibilité à l'insuline dans les cellules musculaires dans des conditions lipotoxiques. Nos résultats montrent que CERT joue un rôle crucial dans les mécanismes conduisant au développement de l'insulinorésistance musculaire puisque sa présence est essentielle pour le maintien d'un trafic normal des CER entre le RE et le golgi. / Obesity and type 2 diabetes are associated with a sedentary lifestyle and a diet rich in fat. Indeed, saturated fatty acids accumulate in non-adipose tissue such as skeletal muscle to generate lipids called ceramides (CER). My thesis project was divided into two parts with the objective to prevent CER to act. We have shown that, depending on the structure of the plasma membrane, CER alter the insulin signaling pathway by targeting PKB, a key insulin signalling protein, via a PKCζ pathway in L6 myotubes and a PP2A pathway in C2C12 myotubes. We also demonstrated that CER affect insulin sensitivity via the PKCζ pathway in human muscle cells. Once CER generated in the endoplasmic reticulum (ER), they are transported to the Golgi by a carrier called CERT to be metabolized into sphingomyelin (SM). Studies have shown that the transformation of CER into SM could be a crucial step to prevent CER to act. In several muscle insulin resistance models, expression of CERT is decreased and we demonstrated the importance of the transport of ceramide from the ER to the Golgi by inhibiting artificially the activity or the expression of CERT. In contrast, overexpression of CERT enhances insulin sensitivity in muscle cells in lipotoxiques conditions. Our results show that CERT plays a crucial role in mechanisms leading to the development of muscle insulin resistance since its presence is essential for maintaining normal traffic of CER between the ER and the Golgi.