O estresse oxidativo e a enzima NADPH oxidase-2 (NOX2) estão associados com a diminuição da massa funcional de células-β em pacientes com diabetes do tipo 2 (DT2). Neste estudo, testamos o papel da NOX2 sobre a glicotoxicidade em células-β. Ilhotas de camundongo C57BL/6J nocautes ou não para NOX2 (NOX2-KO e WT, respectivamente) foram isoladas e cultivadas por até 3 semanas em 10 ou 30 mmol/l de glucose (G10 e G30, respectivamente). A secreção de insulina foi maior nas ilhotas NOX2-KO vs. WT sem apresentar diferenças metabólicas ou do potencial redox da glutationa citosólica (EGSH). O cultivo de ilhotas em G30 aumenta a concentração de H2O2 e a oxidação de tióis no compartimento citosólico, seguido por aumento de apoptose de células-β, mas, preservando a reposta máxima secretória. Estas respostas foram quase idênticas em ambos os tipos de ilhotas. Em conclusão, a NOX2 regula negativamente a secreção de insulina em ilhotas de camundongos C57BL/6J, mas não é um componente crítico para a sobrevivência de células β em um modelo in vitro de glicotoxicidade. / Oxidative stress and NADPH oxidase-2 (NOX2) enzyme are associated to the decline of the functional β-cell mass in type 2 diabetes (T2D). Here, we tested the role of NOX2 on β-cell glucotoxicity. NOX2 knockout (NOX2 KO) and wild type (WT) C57BL/6J mice islets were isolated and cultured up to 3 weeks at 10 or 30 mmol/l glucose concentrations (G10 and G30, respectively). The insulin secretion was higher in NOX2-KO vs. WT islets despite similar metabolic and cytosolic glutathione-redox potential (EGSH) changes. The prolonged culture at G30 increases the H2O2 concentration and cytosolic thiol oxidation, followed by increased βcell apoptosis but preserving maximal secretory response. These responses were almost identical in both types of islets. In conclusion, NOX2 is a negative regulator of insulin secretion in C57BL/6J mouse islets, but is not a critical component for β-cell survival in a model of glucotoxicity in vitro.

O receptor ativado do peroxissomo γ (PPAR-γ) é regulador do metabolismo e diferenciação do tecido adiposo, sendo um alvo conhecido das tiazolidinedionas (TZD), utilizadas para o tratamento do diabetes tipo 2 (DM2). As TZD agem como um agente sensibilizador da ação da insulina nos tecidos periféricos e tem sido especulado que as TZDs podem ter um papel na função da célula , prevenindo perda de massa e melhorando a sua viabilidade a longo prazo. Este efeito seria supostamente mediado pela transcrição de genes que favoreceriam a lipólise, diminuindo o conteúdo intracelular de triglicérides e, portanto, diminuindo a lipotoxicidade. Entretanto, alguns estudos também mostraram efeito nulo ou mesmo deletério das TZDs sobre as ilhotas pancreáticas. Na realidade, o papel de genes-alvo para o PPAR- nas ilhotas pancreáticas é ainda pouco conhecido. Estudamos o perfil de expressão gênica induzido pelo tratamento com Pioglitazona (Pio), uma TZD aprovada e disponível para uso clínico no tratamento do DM2, em ilhotas pancreáticas murídeas em cultura primária, com concentrações normal e suprafisiológica de glicose no meio de cultura. As ilhotas foram obtidas de ratos wistar machos de dois meses de idade e isoladas pelo método do gradiente de Ficoll e então cultivadas em 5,6 mM ou 23 mM de glicose por 24h, sendo tratadas com Pio 10 M ou DMSO 0,1% (veículo). A Pioglitazona foi cedida pela Takeda Farmacêutica, Osaka, Japão. O RNA foi extraído com Trizol e purificado com o kit RNeasy (Qiagen). As amostras foram marcadas e hibridizadas no microarranjo de cDNA Mouse Panchip 13k, usando-se cinco replicatas biológicas diferentes para cada condição. A análise estatística dos dados do microarranjo foi feita com o uso do programa significance analysis of microarrays (SAM) com uso de taxa de descobrimento falso (FDR) de 20%. A análise das vias acometidas foi feita com o Ingenuity Pathway Analysis (www.ingenuity.com). Os resultados de expressão gênica foram confirmados por RT-qPCR. Em concentração de 5,6 mM de glicose no meio de cultura, 101 genes foram modulados pela Pio, sendo 49 regulados para cima, com aumento de sua expressão na presença da droga e 52 genes regulados para baixo. Em 23 mM de glicose, 1.235 genes foram afetados, sendo 621 para cima e 623 para baixo. A comparação entre as duas condições revelou 74 genes que foram modulados em ambas as concentrações de glicose. A análise das vias biológicas alteradas mostrou que genes relacionados ao metabolismo de lípides foram modulados em ambas as concentrações de glicose. Em 23 mM foi ainda significativo o grupo de genes relacionados a ciclo celular e morte celular que tiveram sua expressão modificada pela presença da droga na cultura. Este dado demonstrou que além de seus efeitos conhecidos nos adipócitos, o sensibilizador de insulina Pioglitazona modula a expressão de genes nas ilhotas pancreáticas, especialmente na presença de concentrações suprafisiológicas de glicose, afetando notadamente genes relacionados ao metabolismo lipídico, sendo vários deles ligados a lipogênese, como Srebf1, Scd2 e Fabp4 cujas expressões aumentaram em ambas as concentrações de glicose. Além disso foi observado aumento na expressão de genes com atividade pró-apoptótica como Tnf, Bad, Bax, Caspase4, Fadd e Myc. A Pioglitazona parece induzir um perfil gênico desfavorável em ilhotas pancreáticas mantidas em cultura em concentrações suprafisiológicas de glicose. / Peroxisome proliferator-activator receptor-γ (PPAR-γ) is a target for thiazolidinedione (TZD) antidiabetic drugs and a regulator of adipose tissue differentiation and metabolism. TZD act as an insulin sensitizing agent on peripheral tissues. It has been speculated that TZD could play a role on beta-cell function, preventing loss and improving viability in the long-term. This effect is supposed to be mediated through a potential benefit against lipotoxicity, favouring lypolisis and decreasing intracellular tryglicerides content. Nevertheless some studies also showed a lack or even a potential deleterious effect of TZD on islets. The role of PPAR-γ target genes in pancreatic islets is actually still largely unclear. We studied the gene expression profile induced by the treatment with Pioglitazone (Pio), an approved TZD for T2DM therapy, on rat pancreatic islets primary culture both at normal and supraphysiological glucose medium concentrations. Islets were obtained from 2 month-old, male, wistar rats and isolated through the Ficoll gradient method and then cultured with 5.6 mM or 23 mM of glucose concentration for 24h, being treated with Pio 10 µM or DMSO 0.1% (vehicle). Pioglitazone was provided by Takeda Pharmaceuticals, Osaka, Japan. RNA was extracted with Trizol (Sigma) and purified with RNeasy kit (Qiagen). Samples were labeled and then hybridized on the Mouse PanChip 13k cDNA microarray, using 5 different biological replicates for each test condition. Statistical Analysis of the microarray data was performed using significance analysis of microarrays (SAM) with a false discovery rate of 20%. Pathways assessment was performed through Ingenuity Pathway Analysis (www.ingenuity.com). Gene expression results were confirmed through RT-qPCR. At 5.6 mM glucose 101 genes were modulated by Pio, 49 upregulated and 52 downregulated. At 23 mM, 1,235 genes were affected, 612 upregulated and 623 downregulated. Comparison between both conditions revealed 74 genes that were similarly modulated at both glucose concentrations. Pathway analysis of perturbed genes revealed biologically relevant networks related to lipid metabolism at both glucose medium concentrations. At 23 mM, cell cycle and cell death pathways were significant modulated as well. These data demonstrates that in addition to known effect in adipocytes, the insulin sensitizing agent Pioglitazone modulates gene expression in pancreatic islets, especially in the presence of supraphysiological glucose concentrations, affecting especially lipid metabolism and mechanisms of cell death and cell cycle. Considering the ontology of modulated genes it seems to be a trend towards lypogenesis (increased Srebf1, Scd2 and Fabp4 RNA expressions) with Pio treatment also enhancing the abundance of some genes considered to be pro apoptotic like Tnf, Bad, Bax, Caspase4, Fadd and Myc. Pioglitazone seems to induce a negative gene expression profile in islets cultured at high glucose concentrations.

A secreção de insulina é estimulada pela glicose, porém os ácidos graxos (AG) podem influenciar o processo secretório. A oxidação de AG é importante para a estimulação da secreção de insulina por aumentar o ATP, porém, existem vias dependentes e independentes de ATP. Os AG <font face=\"Symbol\">w-3 interferem em processos fisiológicos e na composição e função da membrana plasmática, promovendo potente ação anti-inflamatória. Considerando a importante relação da NAD(P)H oxidase com a secreção de insulina, o estudo das alterações induzidas pela suplementação com AG <font face=\"Symbol\">w-3 sobre o conteúdo de superóxido (O2<font face=\"Symbol\">&#183;) e a expressão da NAD(P)H oxidase, é importante para a compreensão da fisiologia das células <font face=\"Symbol\">b-pancreáticas. Neste estudo, o grupo suplementado apresentou redução do conteúdo de O2<font face=\"Symbol\">&#183;, redução da expressão das subunidades da NAD(P)H oxidase e aumento na expressão da superóxido dismutase (SOD1 e 2), quando comparado ao grupo controle. Embora desconhecido o mecanismo, este dado é relevante, pois pressupõe melhor regulação do estado redox durante a secreção de insulina. / Insulin secretion is stimulated by glucose (GSIS), but fatty acid (FA) may influence the secretory process. The oxidation of FA is important for the stimulation of insulin secretion by increasing the ATP, although there are dependent and independent ATP pathways. The <font face=\"Symbol\">w-3 FA change physiological processes, and affect the composition and function of the plasma membrane, promote potent anti-inflammatory action. Considering the important relationship of NAD(P)H oxidase with insulin secretion, the study of changes induced by supplementation with <font face=\"Symbol\">w-3 FA on the superoxide (O2<font face=\"Symbol\">&#183;) content, and expression of NAD(P)H oxidase, becomes of great importance for understanding the pancreatic <font face=\"Symbol\">b cells physiology. In this study, the group supplemented with <font face=\"Symbol\">w-3 FA showed a reduction of the O2<font face=\"Symbol\">&#183; content, reduced expression of NAD(P)H oxidase subunits, and increased the expression of the enzyme superoxide dismutase (SOD 1 and 2), compared to control. Although unknown the mechanism, this data is relevant, because it represents better regulation of the redox state during GSIS .

Type 1 diabetes develops through the progressive destruction of the insulin-producing beta-cells. Regeneration or replacement of beta-cells is therefore needed to restore normal glucose homeostasis. Presently, normoglycemia can be achieved by the transplantation of whole pancreas or isolated islets of Langerhans. Islet transplantation can be performed through a simple laparoscopic procedure, but the long-term graft survival is low due to poor revascularization and early cell death. This thesis examined the possibility of using different auxiliary cells (Schwann cells, endothelial progenitor cells, and neural crest stem cells) to improve the engraftment and function of endogenous and transplanted islets. Co-transplantation of Schwann cells with islets improved islet graft function early after transplantation, and caused an increased islet mass at one month posttransplantation. However, the vascular densities of these grafts were decreased, which also related to an impaired graft function. Islet grafts containing endothelial progenitor cells had a superior vascular density, with functional chimeric blood vessels and substantially higher blood perfusion and oxygen tension than control transplants. By culturing and transplanting islets together with neural crest stem cells it was found that islets exposed to these cells had a higher beta-cell proliferation compared with control islets. At one month posttransplantation, the grafts with neural crest stem cells also had a superior vascular- and neural density. The potential of intracardially injected neural crest stem cells to home to the pancreas and ameliorate hyperglycemia in diabetic mice was investigated. During a three-week period after such cell treatment blood glucose concentrations decreased, but were not fully normalized. Neural crest stem cells were present in more than 10% of the pancreatic islets at two days postinjection, at which time the beta-cell proliferation was markedly increased when compared with islets of saline-treated diabetic animals. Three weeks later, a doubled beta-cell mass was observed in animals receiving neural crest stem cells. In summary, islets can easily be transplanted together with different auxiliary cells. Some of these cells provide the possibility of improving vascular- and neural engraftment, as well as beta-cell growth and survival. Systemic administration of neural crest stem cells holds the potential of regenerating the endogenous beta-cells.

Orientador: Everardo Magalhães Carneiro / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-18T10:36:45Z (GMT). No. of bitstreams: 1 Silva_PriscillaMunizRibeiroda_D.pdf: 2752788 bytes, checksum: a2b912ed65ab26f33708f44d094db90a (MD5) Previous issue date: 2011 / Resumo: A glutamato desidrogenase (GDH) é uma enzima mitochondrial que cataliza a reação reversível de glutamato a ?-cetoglutarato. Nas ilhotas pancreáticas, está associada com a secreção de insulina por aumentar a concentração de ATP. Ratos alimentados com dieta hipoprotéica apresentam secreção de insulina diminuída. A suplementação com leucina (LEU) aumenta a secreção de insulina em resposta a agentes insulinotrópicos. O presente estudo investigou a influência da suplementação com LEU na expressão da GDH e seu envolvimento com a secreção de insulina em ratos desnutridos e suplementados com LEU. Ratos machos foram alimentados com dietas normo- (17%, NP) ou hipoprotéicas (6%, LP) por oito semanas. Após, foram divididos e suplementados com LEU (1,5%) na água de beber (NPL e LPL) pelas quatro semanas seguintes. O conteúdo protéico de GDH no cérebro, fígado, rim e músculo esquelético não diferiu entre os grupos. Nas ilhotas LP, a expressão da GDH estava diminuída e a suplementação com LEU aumentou a expressão de RNAm restaurando o conteúdo protéico a valores similares a NP. A secreção de insulina estimulada por agentes insulinotrópicos ou inibidores, combinados ou não, estava diminuída em ilhotas LP comparada com NP. A suplementação com LEU aumentou a secreção de insulina a valores similares a NP, exceto quando as ilhotas LPL foram incubadas com EGCG. Ilhotas LP tiveram diminuição na [Ca2+]i quando expostas a GLN+BCH. A suplementação com LEU restaurou esses parâmetros aos valores de NP. Frente a esses resultados, podemos concluir que a diminuição na expressão da GDH induzida pela dieta LP foi central ao pâncreas endócrino e está associada à redução da secreção de insulina observada nas ilhotas LP. A suplementação com LEU foi capaz de restaurar a expressão da GDH, contribuindo para o aumento da secreção de insulina observado nas ilhotas LPL. Além disso, a GDH pode, ainda, estar associada com a secreção de insulina pelo acoplamento estímulo/secreção via regulação da [Ca2+]i / Abstract: Glutamate dehydrogenase (GDH) is a mitochondrial matrix enzyme that catalyzes the reversible reaction of glutamate to ?-ketoglutarate. In the pancreatic islets, this enzyme is associated with insulin secretion by augmenting ATP levels. Protein malnourished rats displayed reduced insulin secretion. Leucine (LEU) supplementation augments the insulin secretion response to insulinotropic agents. The present study investigated the influence of LEU supplementation on GDH expression and its involvement with insulin secretion in malnourished rats supplemented with LEU. Male rats were fed normal- (17%, NP) or low-protein diet (6%, LP) for eight weeks. Half of rats of each group were supplemented with LEU (1.5%) in drinking water for the following four weeks (NPL and LPL groups). GDH protein content in brain, liver, kidney and skeletal muscles was not different in any group. GDH RNAm and protein content was reduced in LP islets and LEU supplementation augmented RNAm expression restoring protein content similar to NP. Insulin secretion was reduced in LP islets compared with NP when stimulated by insulinotropics agents or inhybitors, combinaned or not. LEU supplementation augmented insulin secretion to similar values as NP, an effect that was blunted when LPL islets were incubated with EGCG. LP islets showed lower [Ca2+]i when exposed to GLN+BCH. LEU supplementation augmented these patterns similar to NP. Taken together, we may conclude that diminution in GDH expression induced by LP diet was central to endocrine pancreas and was associated with reduced insulin secretion observed in LP rats. LEU supplementation was able to restore GDH expression and it was capable to restore insulin secretion via GDH restoration. Yet, GDH may contribute to insulin secretion via Ca2+ regulation stimulus/secretion coupling / Doutorado / Fisiologia / Doutor em Biologia Funcional e Molecular

<p>Akrilamid&nbsp; je toksična hemijska supst anca koja je već dugi niz godina prisutna u životnoj sredini,&nbsp; jer se kao važan monomer koristi u različite industrijske i laboratorijske svrhe. U poslednjih petnaest godina, akrilamid je postao posebno zanimljiv za &scaron;ire naučne krugove jer&nbsp; se pokazalo da&nbsp; se&nbsp; nalazi&nbsp; i u&nbsp; hrani&nbsp; biljnog porekla, posebno hrani bogatoj skrobom, koja se priprema pečenjem ili prženjem na temperaturama vi&scaron;im od 120&deg;C.&nbsp; Do sada ustanovljeni negativni zdravstveni efekti akrilamida su veoma raznovrsni i mogu biti rezultat delovanja samog&nbsp; akrilamida ili delovanja njegovog metabolita glicidamida koji nastaje&nbsp; in vivo&nbsp; kada se jedan deo molekula akrilamida metaboli&scaron;e oksigenacijom dvostruke veze pomoću enzima citohrom P450 2E1 (CYP2E1). Akrilamid je supstanca koja ima dokazan negativan efekat&nbsp; na organske sisteme kod ljudi i životinja, i koja je svrstana u moguće humane karcinogene. Negativan efekat akrilamida na egzokrini pankreas je poznat, ali o mogućim efektima akrilamida na endokrini pankreas se i dalje veoma malo zna. Ima puno dokaza koji&nbsp; ukazuju na to da akrilamid ima citotoksični efekat koji se&nbsp; manifestuje kroz uticaj na redoks-status ćelija i dovodi do promena u vrednostima biomarkera oksidativnog i nitrozativnog stresa, kao i u aktivnosti antioksidativnih enzima. Pankreas&nbsp; je&nbsp; jedan od ciljnih&nbsp; organa za delovanje akrilamida te je&nbsp; glavni predmet istraživanja&nbsp; doktorske teze&nbsp; bio proučavanje potencijalnog efekta akrilamida na endokrini pankreas pacova.&nbsp; Ispitivanje je vr&scaron;eno na 3&nbsp; eksperimentalne grupe&nbsp; juvenilnih&nbsp; mužjaka pacova soja Wistar,&nbsp; od kojih je&nbsp; jedna grupa bila kontrolna, dok su dve bile tretirane&nbsp; sa akrilamidom u dozama od 25 mg/kg tm i 50 mg/kg tm,&nbsp; 5 dana nedeljno,&nbsp; tokom 3 nedelje. Po isteku tretmana,&nbsp; nakon dekapitacije, kompletno tkivo pankreasa&nbsp; je&nbsp; fiksirano u 10% rastvoru formalina&nbsp; tokom&nbsp; 24&nbsp; h i obrađeno prema&nbsp; standardnoj proceduri za kalupljenje u parafinu.&nbsp; Parafinski kalupi su sečeni na serijske preseke debljine 5 &micro;m, nakon čega su bojeni&nbsp; histohemijskom i imunohistohemijskim metodama.&nbsp; Kod eksperimentalnih grupa posmatrane&nbsp; su&nbsp; histolo&scaron;ke promene na endokrinom pankreasu, sa akcentom na &alpha;-&nbsp; i &beta;-ćelije.&nbsp; Takođe, posmatrana je&nbsp; i&nbsp; ekspresija&nbsp; hormona insulina i glukagona, enzima inducibilne azot -oksi d&nbsp; sintetaze (iNOS) i&nbsp; CYP2E1,&nbsp; kao&nbsp; i ekspresija&nbsp;&nbsp; antioksidativnih enzima&nbsp; katalaza&nbsp; (CAT) i superoksid dismut aza 1 i 2&nbsp; (SOD1 i SOD2)&nbsp; u ćelijama Langerhansovih ostrvaca. Potencijalna promena u funkcionalnosti &beta;-ćelija je ispitana i kroz analizu nivoa glukoze u serumu pacova tretiranih sa akrilamidom.<br />Budući da &beta;-ćelije čine 80% ćelija koje grade Langerhansova ostrvca pankreasa,&nbsp; pored in vivo&nbsp; eksperimenata, ispitana&nbsp; je&nbsp; i toksičnost akrilamida na&nbsp; Rin-5F ćelijsku liniju insulinoma &beta;-ćelija pacova u in vitro uslovima. Glavni cilj in vitro&nbsp; istraživanja je bio&nbsp; da se&nbsp; ispita&nbsp; uticaj&nbsp; rastućih&nbsp; koncentracija akrilamida na preživljavanje tretiranih&nbsp; Rin-5F&nbsp; ćelija, ali i efekat IC₅₀&nbsp; koncentracije ove supstance primenjene&nbsp; tokom&nbsp; različitih vremenskih intervala&nbsp; (0,5, 1, 3, 6, 12 i 24 h)&nbsp; na pojavu oksidativnog i nitrozativnog stresa. Redoks-status Rin-5F ćelija tretiranih&nbsp; sa akrilamidom je ispitan preko analize prisustva biomarkera oksidativnog i nitrozativnog stresa, akrivnosti CAT i ukupne SOD, kao i promene u ekspresiji gena za CAT, SOD1, SOD2&nbsp;&nbsp; i iNOS.&nbsp; Pored toga, analiziran je i efekat istog tretmana na&nbsp; ekspresiju gena za insulin, CYP2E1, Bax i Bcl-2. U okviru teze je pokazano da akrilamid ne dovodi do&nbsp; značajnih promena u histolo&scaron;koj građi, dijametru i broju Langerhansovih ostrvaca&nbsp; kod&nbsp; tretiranih životinja.&nbsp; Primena stereolo&scaron;kih metoda&nbsp; je&nbsp; ukazala&nbsp; na mikrostrukturne promene na&nbsp; endokrinom pankreasu na nivou &alpha;-&nbsp; i &beta;-ćelija. U ovoj tezi je po prvi put pokazano da tretman akrilamidom negativno utiče na broj i povr&scaron;inu &beta;-ćelija pankreasa.&nbsp; U tezi je, takođe,&nbsp; pokazan&nbsp; značajan dozno-zavisni pad u prisustvu insulina u &beta;-ćelijama&nbsp;&nbsp; pankreasa. Uprkos&nbsp; tome, kod&nbsp; akrilamidom tretiranih&nbsp; životinja&nbsp; nije konstatovana&nbsp; promena&nbsp; u&nbsp; koncentraciji serumske glukoze.&nbsp; U&nbsp; ovoj tezi je pokazano da tretman akrilamidom dovodi do&nbsp;&nbsp; statistički značajnog porasta&nbsp; u broju &alpha;-ćelija&nbsp; kod životinja koje su primale nižu dozu tretmana, dok se&nbsp; broj &alpha;-ćelija&nbsp; kod životinja koje su primale vi&scaron;u dozu tretmana&nbsp; ne razlikuje značajno od kontrole.&nbsp; Tretman akrilamidom je doveo do značajnog&nbsp; porasta u količini&nbsp;&nbsp; prisutnog glukagona&nbsp; u &alpha;-ćelijama pankreasa.<br />Tretman akrilamidom nije doveo do značajne promene u ekspresiji CAT, SOD1 i SOD2 u ćelijama Langerhansovih ostrvaca.&nbsp; Kod&nbsp; tretiranih životinja&nbsp; do&scaron;lo do značajnog dozno-zavisnog porasta&nbsp; u ekspresiji&nbsp; enzima iNOS,&nbsp; dok je ekspresija&nbsp; CYP2E1 značajno dozno-zavisno opala&nbsp; nakon tretmana. U&nbsp; tezi je pokazano da tretman akrilamidom negativno utiče na vijabilnost Rin-5F ćelija, i utvrđeno je da IC50&nbsp; koncentracija akrilamida za Rin-5F ćelije iznosi 10 mM.&nbsp; Rezultati teze pokazuju da tretman akrilamidom u IC₅₀&nbsp; koncentraciji u Rin-5F ćelijskoj liniji značajno povećava nivo malondialdehida (MDA) nakon tretmana u trajanju od 1, 12 i 24 h.&nbsp; Isti tretman&nbsp; značajno smanjuje nivo redukovanog GSH nakon tretmana od 1, 3, 6, 12 i<br />24 h, kao i nivo slobodnih&nbsp; &ndash;SH grupa nakon tretmana od 3 i 6 h. Tretman akrilamidom u IC_50&nbsp; koncentraciji signifikantno pojačava aktivnost CAT nakon tretmana od 1 h, dok tretman u trajanju od 12 h značajno smanjuje aktivnost ovog enzima. Ovaj tretman smanjuje aktivnost SOD nakon 1, 12 i 24 h, dok&nbsp; tretman u trajanju od 6 h značajno pojačava aktivnost enzima SOD.&nbsp; U tezi je, takođe, pokazan i veoma značajan porast&nbsp; u nivou prisutnih nitrita,&nbsp; koji&nbsp; je direktno proporcionalan&nbsp; sa nivoom azot-oksida i nivoom akivnosti enzima iNOS.&nbsp; Ovaj&nbsp; nalaz ukazuje na potencijalnu pojavu nitrozati vnog stresa u akrilamidom-tretiranim Rin-5F ćelijama.&nbsp; U&nbsp; tezi je po prvi put pokazano da tretman&nbsp; akrilamidom dovodi do&nbsp; značajnih&nbsp; varijacija&nbsp; u transkripciji gena za iNOS, SOD1, SOD2,&nbsp; CAT,&nbsp; CYP2E1,&nbsp; Bax i Bcl-2 u tretiranim Rin-5F ćelijama, dok isti tretman ne dovodi do&nbsp; promene nivoa&nbsp; transkripcije gena za insulin.&nbsp; Tretman akrilamidom u koncentraciji od 10<br />mM tokom rastućih vremenskih perioda dovodi do porasta u relativnoj količini iRNK<br />gena za iNOS u svim tačkama tretmana, do porasta&nbsp; nivoa&nbsp; iRNK za SOD1 i SOD2 nakon tretmana od 12 i 24 h, kao i do porasta&nbsp; količine&nbsp; iRNK za CAT nakon tretmana od 3 h.&nbsp; U&nbsp; tezi je pokazano&nbsp; i&nbsp; da akrilamid&nbsp; izaziva&nbsp; promene&nbsp; u sintezi&nbsp; iRNK&nbsp; za enzim&nbsp; CYP2E1&nbsp; koji je&nbsp; posebno značajan u kontekstu detoksikacije ove toksične supstance.&nbsp; Porast u transkripciji gena za&nbsp; CYP2E1&nbsp; je uočen&nbsp; nakon tretmana u trajanju od 0,5 i 1 h, dok je&nbsp; do smanjenja transkripcije&nbsp; do&scaron;lo&nbsp; nakon tretmana od 12&nbsp; i 24&nbsp; h.&nbsp; Tretman akrilamidom u koncentraciji od&nbsp; 10 mM tokom rastućih vremenskih perioda dovodi do porasta u relativnoj količini iRNK&nbsp; gena za Bax u svim tačkama tretmana, i do porasta u transkripciji gena za Bcl-2 nakon tretmana od 0,5, 1 i 3 h.<br />Sumirajući&nbsp; sve&nbsp; rezultate&nbsp; ove teze,&nbsp; moze se zaključiti&nbsp; da je endokrini pankreas&nbsp; jedno od&nbsp; ciljnih tkiva, na koje akrilamid ostvaruje vi&scaron;estruki negativni uticaj.</p> / <p>Acrylamide is a toxic chemical used as an important monomer for various industrial and laboratory purposes, which makes it highly present in the environment. In the last fifteen years, acrylamide has become especially interesting for wider scientific circles when it was found in staple foodstuff rich in starch, prepared at temperatures higher than 120&deg;C. The established negative health effects of acrylamide are very diverse and can be the result of the acrylamide action itself or the action of its metabolite glycidamide that occurs in vivo, when acrylamide molecule is metabolized via oxygenation of the double bond by the cytochrome P450 2E1 (CYP2E1). Acrylamide is a substance with a proven adverse effect on humans and animals, and it is classified as a possible human carcinogen. The negative effect of acrylamide on the exocrine pancreas has already been recognized, but the possible effects of acrylamide&nbsp; on endocrine pancreas are still mostly undetermined. There is a significant amount of evidence to suggest that acrylamide exerts a cytotoxic effect which manifests through the changes in level of oxidative and nitrosative stress biomarkers, as well as in the activity of antioxidant enzymes. Since, pancreas is one of the target organs for acrylamide, the main subject of doctoral thesis was to investigate the potential effect of acrylamide on the rat endocrine pancreas. The investigation was conducted on 3 experimental groups of juvenile male Wistar rats, of which one group was the control group, while two groups were treated with acrylamide at doses of 25 mg/kg bw and 50 mg/kg bw, 5 days a week, during 3 weeks. After termination of the treatment, decapitation was performed, and the complete pancreatic tissue was fixed in a 10% formalin solution for 24 h and treated according to the standard paraffin embedding procedure. Paraffin molds were cut into 5 &mu;m thick serial sections, after which they were stained with histochemical and immunohistochemical methods. Histological changes ofthe endocrine pancreas, with the emphasis on &alpha;- and &beta;-cells, were examined in three experimental groups of rats. In addition, the expression of insulin and glucagon hormone, the inducible nitric oxide synthase (iNOS) and CYP2E1 enzymes, and the expression of antioxidative enzymes catalase (CAT) and superoxide dismutases 1 and 2&nbsp; (SOD1 and SOD2) in the islets of Langerhans were also investigated. A potential change in the functionality of &beta;-cells was also examined by analyzing glucose level in the serum of rats treated with acrylamide. In pancreatic islets of Langerhans the majority of cells (&gt;80%) are &beta;-cells. Therefore, in addition to in vivo experiments, the toxicity of acrylamide was examined in vitro on rat insulinoma Rin-5F cell line.The main goal of in vitro research was to investigate the impact of increasing acrylamide concentrations on the viability of treated Rin-5F cells, and also to examine whether IC50 concentration of this substance, applied at different intervals of time (0.5, 1, 3, 6, 12 and 24 h), induce oxidative and nitrosative stress. Redox-status of Rin-5F cells treated with acrylamide was examined by analyzing oxidative and nitrosative stress biomarkers, CAT and total SOD activity, as well as changes in the expression of the CAT, SOD1, SOD2 and iNOS. In addition, the effect of the same treatment on the transcription of the insulin, CYP2E1, Bax and Bcl-2 gene was analyzed.The results of the thesis showed that acrylamide treatment does not lead to significant changes in the histological structure, diameter and number of islets of Langerhans of treated animals. Application of stereological methods indicated microstructural changes of &alpha;- and &beta;-cells ofendocrine pancreas. It has been shown for the first time that treatment with acrylamide negatively affects the number and surface area of pancreatic &beta;-cells. In addition, a significant dose-dependent decline in the amount of insulin in pancreatic &beta;-cells was also demonstrated. However, no change in serum glucose level was observed in treated animals. Acrylamide treatment led to a statistically significant increase in the number of &alpha;-cells in animals receiving a lower dose of treatment, while the number of &alpha;-cells in animals receiving a higher dose of treatment did not differ significantly from the control. Treatment with acrylamide led to a significant increase in the amount of the glucagon in &alpha;-cells. Treatment with acrylamide did not cause a significant change in the expression of CAT, SOD1 and SOD2 in islets of Langerhans. However, there was a significant dosedependent increase in the&nbsp; expression of iNOS enzyme, whereas expression of CYP2E1 significantly decreased in dose-dependent manner in treated animals. Results of the thesis showed that acrylamide exerts a negative effect on the viability of Rin-5F cell line. It has been established that the IC50 concentration of acrylamide for the Rin-5F cell line is 10 mM. The results of the thesis indicate that treatment of Rin-5F cell line with IC50 concentration of acrylamide for 1, 12, and 24 h significantly increased the level of malondialdehyde (MDA). Exposure to acrylamide for 1, 3, 6, 12 and 24 h significantly decreased the level of reduced GSH, while the level of free -SH groups was reduced after 3 and 6 h of acrylamide treatments. Treatment with IC50 concentration of acrylamide significantly enhanced CAT activity after 1 h of acrylamide exposure, while 12 h exposure significantly reduced the activity of this enzyme. Application of acrylamide reduced SOD activity after 1, 12, and 24 h exposure, while 6 h exposure significantly increased the activity of SOD enzymes. Results of the thesis also showed a very significant increase of the nitrite level, which is directly proportional to the level of nitrogen oxide (NO) and the level of the iNOS activity. This finding points to the potential occurrence of nitrosative stress in acrylamide-treated Rin-5F cells. It has been shown for the first time that acrylamide treatment leads to significant variations in transcription of iNOS, SOD1, SOD2, CAT, CYP2E1, Bax and Bcl-2 genes in treated Rin-5F cells, while the same treatment does not affect transcription of the insulin gene. Treatment with acrylamide at a concentration of 10 mM for increasing periods of time leads to an increase in the relative amount of the iNOS gene iRNA at all treatment points. Twelve and and 24 h of acrylamide exposure increased the transcription of the SOD1 and SOD2 genes. Transcription of CAT gene was increased after 3 h&nbsp; ofacrylamide exposure. Furthermore, it has been shown that acrylamide treatment leads to variations in the mRNA synthesis of CYP2E1 gene, which is particularly significant in the context of detoxification of this toxic substance. An increase in the transcription ofthe CYP2E1&nbsp; gene was observed after 0.5 and 1 h of acrylamide exposure, while the reduction of&nbsp; transcription occurred after 12 and 24 h of acrylamide exposure. The treatment with 10 mM acrylamide has led to an increase of the transcription of the Bax gene at all treatment points, and also to an increase of transcription of the Bcl-2 gene after of 0.5, 1, and 3 h of acrylamide exposure. Summarizing all the results of this thesis, it can be concluded that the endocrine pancreas&nbsp; is one of the target tissues of acrylamide, to which this substance exerts a multiple adverse effects.</p>

Les îlots de Langerhans, principalement composés de cellules bêta sécrétant l’insuline, jouent un rôle majeur dans l’homéostasie glucidique grâce à leur sécrétion hormonale finement régulée. Dans un contexte d’insulino-résistance associée à l’obésité, la masse fonctionnelle des cellules bêta pancréatiques augmente, en partie grâce à une prolifération accrue. Le diabète de type 2 survient lorsque les mécanismes de compensation échouent et que la sécrétion d’insuline devient insuffisante. Par conséquent, augmenter la prolifération des cellules bêta a été proposée comme approche thérapeutique afin de retarder l’apparition du diabète de type 2. Parmi les différents facteurs pouvant moduler la prolifération des cellules bêta, les nutriments, en particulier le glucose et les acides gras, jouent un rôle important et plusieurs études chez le rongeur montrent que les nutriments augmentent la prolifération et la masse des cellules bêta avant l’apparition de l’insulino-résistance. De plus, des travaux de notre laboratoire ont montré que l’infusion d’un mélange d’acide gras, le ClinOleic (65% oléate, 20% linoléate et 15% palmitate) et de glucose provoquait une augmentation marquée de la prolifération des cellules bêta chez le rat. L’objectif de cette thèse est donc d’évaluer les mécanismes par lesquels les acides gras stimulent la prolifération des cellules bêta. Dans un premier article, seul l’oléate, parmi plusieurs acides gras testé, a démonté un effet significatif sur l’augmentation de la prolifération des cellules bêta en présence de glucose ex vivo. La prolifération induite par l’oléate nécessite la formation de sphingolipides à très longue chaîne monoinsaturée, tandis que la perturbation de leur synthèse provoque une diminution de la réponse proliférative. Dans une seconde étude, l’analyse par séquençage d’ARN sur cellules uniques a mis en évidence le rôle important des espèces réactives de l’oxygène, des peroxyrédoxines et du proto-oncogène MYC dans le processus prolifératif des cellules bêta induit par l’oléate. Dans l’ensemble, les travaux présentés dans cette thèse apportent un éclairage nouveau sur le potentiel prolifératif encore énigmatique des cellules bêta pancréatiques et soulignent le rôle des sphingolipides et des espèces réactives de l’oxygène dans ce processus. / The islets of Langerhans, mainly composed of insulin-secreting beta cells, plays a major role in glucose homeostasis due to their finely regulated hormone secretion. In a context of insulin resistance associated with obesity, the functional mass of pancreatic beta cells increases, in part due to increased proliferation. Type 2 diabetes occurs when these compensatory mechanisms fail and insulin secretion becomes insufficient. Therefore, increasing beta cell proliferation has been proposed as a therapeutic approach to delay the onset of type 2 diabetes. Among the various factors that can modulate the proliferation of beta cells, nutrients, in particular glucose and fatty acids, play an important role, and several studies in rodents show that nutrients increase beta-cell proliferation before insulin resistance can be detected. Previous work from our laboratory has shown that infusion of the fatty-acid mixture ClinOleic (65% oleate, 20% linoleate and 15% palmitate) in the presence of glucose markedly increases beta-cell proliferation in rats. The objective of this thesis is to evaluate the underlying mechanisms by which fatty acids stimulate beta-cell proliferation. In a first study, among several fatty acids tested, only oleate increased beta cell proliferation in presence of glucose ex vivo. Oleate-induced beta-cell proliferation requires the formation of monounsaturated very long chain sphingolipids, while blockade of their biosynthesis dampens the proliferative response. In a second study, single-cell RNA sequencing analysis highlighted the role of reactive oxygen species, peroxiredoxins, and the proto-oncogene MYC in oleate-induced beta cell proliferation. Overall, the work presented in this thesis sheds new light on the enigmatic proliferative potential of pancreatic beta cells and identifies a role for sphingolipids and reactive oxygen species in this process.

Le diabète de type 2 (DT2) résulte d’une résistance à l’insuline par les tissus périphériques et par un défaut de sécrétion de l’insuline par les cellules β-pancréatiques. Au fil du temps, la compensation des îlots de cellules β pour la résistance à l’insuline échoue et entraine par conséquent une baisse progressive de la fonction des cellules β. Plusieurs facteurs peuvent contribuer à la compensation de la cellule β. Toutefois, la compréhension des mécanismes cellulaires et moléculaires sous-jacents à la compensation de la masse de la cellule β reste à ce jour inconnue. Le but de ce mémoire était d’identifier précisément quel mécanisme pouvait amener à la compensation de la cellule β en réponse à un excès de nutriments et plus précisément à l’augmentation de sa prolifération et de sa masse. Ainsi, avec l’augmentation de la résistance à l’insuline et des facteurs circulants chez les rats de six mois perfusés avec du glucose et de l’intralipide, l’hypothèse a été émise et confirmée lors de notre étude que le facteur de croissance HB-EGF active le récepteur de l’EGF et des voies de signalisations subséquentes telles que mTOR et FoxM1 impliquées dans la prolifération de la cellule β-pancréatique. Collectivement, ces résultats nous permettent de mieux comprendre les mécanismes moléculaires impliqués dans la compensation de la masse de la cellule β dans un état de résistance à l’insuline et peuvent servir de nouvelles approches thérapeutiques pour prévenir ou ralentir le développement du DT2. / Type 2 diabetes (T2D) results from insulin resistance in peripheral tissues and impaired insulin secretion from the pancreatic β-cell. Over the time, compensation of the β cell islets for insulin resistance fails, and therefore leads to a gradual decline in β-cell function. Several factors may contribute to β-cell compensation. However, the cellular and molecular mechanisms underlying β-cell compensation remain unknown. The purpose of this thesis was to identify what mechanism could lead to β cell compensation in response to nutrients excess and specifically the increase in proliferation and β-cell mass. Thus, with increasing insulin resistance and circulating factors in the 6 month rats infused with glucose + intralipid, the hypothesis was made and confirmed in our study that the growth factor HB-EGF would activate the EGF receptor, and subsequent signaling pathways such as mTOR and FoxM1, both involved in the proliferation of the pancreatic beta-cell. Collectively, these results allow us to understand better the molecular mechanisms involved in the β cell compensation in the insulin resistance state and may serve as a potential new therapeutic approach to prevent or delay T2D development.

L’amyloïdose, une maladie progressive et incurable, implique une vaste panoplie de pathologies et de pathogénèses, qui est expliquée par la grande variabilité biologique et structurale des protéines responsables de la formation des dépôts d’amyloïde. L’amyline (polypeptide amyloïde des îlots pancréatiques, IAPP) est une protéine très susceptible de subir des changements de conformation impliquant les feuillets bêta et conférant aussi des propriétés physicochimiques distinctes. Cette protéine prend alors une forme fibrillaire et se dépose dans les îlots de Langerhans chez les humains atteints de diabète de type 2 ou d’insulinome. Ces dépôts d’amyloïde pancréatique (AIAPP) ont été décrits chez certaines espèces animales telles que les félins domestiques, les grands félins, le raton laveur et les primates non humains. La formation de dépôts d’amyloïde contribue à la pathogénèse du diabète de type 2, mais les mécanismes qui induisent la conversion de l’amyline (IAPP) en amyloïde (AIAPP) ne sont pas complètement compris. Les hypothèses du projet sont que certaines variations présentes dans les séquences peptidiques de l’IAPP provenant de différentes espèces animales jouent un rôle critique pour la formation de fibrilles et que plusieurs composés chimiques aromatiques/phénoliques sont capables d’abroger la formation de dépôts d’amyloïde. Le projet de recherche consiste donc à caractériser la propension des différentes isoformes animales d’IAPP à former de l’amyloïde in vitro afin d’identifier les acides aminés jouant un rôle clé dans cette transformation structurale et ultimement d’inhiber la formation d’amyloïde pancréatique. Le projet se divise en deux volets principaux. Le premier consiste à identifier les différentes séquences peptidiques de l’IAPP retrouvées chez les espèces animales. L’objectif est d’identifier les acides aminés jouant un rôle clé dans la formation d’amyloïde. Le gène de l’IAPP a été séquencé chez plus d’une quarantaine d’espèces. Le potentiel d’agrégation des séquences obtenues a été simulé à l’aide d’outils bioinformatique. Une librairie de 23 peptides a été commandée afin de procéder à des analyses physicochimiques in vitro permettant d’évaluer le potentiel amyloïdogénique (test fluorimétrique à la thioflavine T, essai de liaison au rouge Congo, dichroïsme circulaire, microscopie électronique à transmission) et cytotoxique (sur une lignée cellulaire provenant d’insulinome : INS-1). Les analyses effectuées à partir de la librairie constituée de 23 peptides ont permis d’identifier trois séquences ne formant pas d’amyloïde et qui proviennent des espèces animales suivantes : le tamarin lion doré (Leontopithecus rosalia), le grand dauphin (Tursiops truncatus) et l’alpaga (Vicugna pacos). Un site potentiellement critique est le segment 8-20 présentant le motif NFLVH qui ne forme plus d’amyloïde lorsqu’il est remplacé par le motif DFLGR ou KFLIR. Les acides aminés 29P, 14K et 18R sont également impliqués dans l’inhibition de la transformation structurale en fibrille. La dernière partie du projet consiste à inhiber la formation de l’amyloïde en utilisant des composés chimiques commercialisés (hypoglycémiants, anti-inflammatoires non stéroïdiens) ou nouvellement synthétisés dans notre laboratoire (les aryles éthyles urées). Un criblage d’une soixantaine de composés chimiques a été conduit dans cette étude. Leur efficacité a été testée sur l’IAPP humaine, qui possède un fort potentiel amyloïdogénique. Les techniques utilisées sont les mêmes que celles exploitées précédemment. L’essai de liaison croisée photo-induite ("photo-induced cross-linking of unmodified proteins", PICUP) a été réalisé afin d’étudier les formes intermédiaires (monomères, oligomères). Un total de 11 composés chimiques a démontré un potentiel à inhiber l’agrégation des fibrilles. Pour la classe des hypoglycémiants, le glyburide, le répaglinide et la troglitazone ont montré l’activité thérapeutique la plus élevée pour retarder et réduire la formation de fibrilles. Les anti-inflammatoires antiamyloïdogènes actifs incluaient le diclofenac, le méloxicam, le phénylbutazone, le sulindac et le ténoxicam. Les aryles étyles urées les plus intéressantes étaient la EU-362 et la EU-418. Tous ces composés ont conféré une protection cellulaire contre l’activité cytotoxique des fibrilles. Les molécules actives possèdent des éléments structuraux communs tels des substituants donneurs d’électrons (alcool, amine, halogène) sur un noyau benzène. En conclusion, ce projet de recherche a permis de caractériser l’IAPP chez diverses espèces animales, dont plusieurs chez lesquelles elle n’avait pas encore été décrite, de déterminer les sites jouant un rôle clé dans sa transformation en amyloïde et, ultimement, de tester le potentiel thérapeutique de nouveaux agents antiamyloïdogènes dans le diabète de type 2. Nous espérons que ce projet ouvrira ainsi la porte à de nouvelles stratégies de traitement. / Amyloidosis is a progressive and, as of now, incurable disease caused by the deposition of insoluble proteins. Amyloid research over the past decades focused on the characterization of the substantive biological variability of amyloid deposits. Amyloidosis encompasses a diversity of pathological manifestations, explained by the diversity of underlying causal proteins. In the pancreas of susceptible species, islet amyloid polypeptide (IAPP) is a precursor for an amyloid protein (AIAPP), which has a characteristic fibrillar structure and resistance to physical agents. This folded protein deposits in the islets of Langerhans of patients with type 2 diabetes or islet cell tumors (insulinoma). Amyloid deposits have also been well characterized, anatomically, in feline and non-human primate species. Amyloid fibril formation contributes to the pathogenesis of diabetes mellitus but the precise pathophysiologic factors involved in the fibrillization of IAPP as well as resultant islet injury remain to be elucidated. Further understanding of the causative factors in the fibrillogenesis of IAPP will be requisite in the development of therapeutic strategies to disrupt the amyloidosis process. This project hypothesizes that the specific variations found in IAPP peptide sequences among different animal species are critical for IAPP fibrillization. Also, some aromatic/polyphenolic compounds can abrogate fibrillization. The main objective forms the basis for development of new therapeutic tactics to impede amyloid formation and associated cellular injury. Thus this project has two specific aims. The first specific aim was to identify critical variations in IAPP amino acid sequences from different animal species and to assess their amyloidogenic potential. To accomplish this, the IAPP gene was isolated and sequenced from paraffin-embedded tissues from various animals (40 species). The aggregation potency was assessed for each sequence using in silico analysis. A library of 23 peptides was prepared from sequences that were distinctly different and their amyloidogenic potential was assessed in vitro using physicochemical analysis (thioflavin-T assay, Congo red binding assay, far-UV circular dichroism, transmission electron microscopy) and cytotoxicity assays (insulinoma cell line INS-1). Among this peptide library, three were non-amyloidogenic and corresponded to the following animal species: golden lion tamarin (Leontopithecus rosalia), commun bottlenose dolphin (Tursiops truncates) and alpaca (Vicugna pacos). Segment 8-20 of the peptide was critical for amyloid formation and the substitution of the NFLVH motif found in this region by a DFLGR or KFLIR motif impeded fibrillization. The amino acids 29P, 14K and 18R were also demonstrated to abrogate fibril formation. The second objective consisted in abrogating IAPP fibrillogenesis using conceptualized aromatic/polyphenolic structures, specifically hypoglycemic, non-steroidal anti-inflammatory and aryl ethyl urea agents. This part of the project involved molecular screening of more than 60 compounds. Their efficacy at inhibiting amyloid formation was assessed in vitro on human IAPP, which exhibits the highest amyloidogenic potential. Techniques included the above-mentioned methods, with the addition of photo-induced cross-linking of unmodified proteins (PICUP). A total of 11 compounds showed potential in abrogating IAPP aggregation. Among the hypoglycemic agents evaluated, glyburide, repaglinide and troglitazone showed the highest potency in reducing fibril formation. The NSAIDs that displayed anti-amyloidogenic activity were diclofenac, meloxicam, phenylbutazone, sulindac and tenoxicam. EU-362 and EU-418 were the hit compounds resulting from the screening of the aryl ethyl urea (EU) class. Additionally, these anti-amyloidogenic molecules conferred a protection against fibril cytotoxicity. All of the active molecules bear a commun motif composed of benzene ring with electron donor moieties, such as alcohol, amine or halide. To conclude, this project characterized IAPP in several animal species in which it has not been previously described and improves our understanding of the amyloidogenesis process. Moreover, the therapeutic potential of hypoglycemic, non-steroidal anti-inflammatory and aryl ethyl ureas agents as anti-amyloidogenic compounds was evaluated. It is conceivable that the additional information hereby gained on the regulation of amyloidogenesis may point towards new therapeutic strategies for diabetic patients.

Diabetes mellitus tipo 1 resulta da produção insuficiente ou da ausência de insulina, decorrente da destruição de células &#946;, por mecanismo auto-imune. O tratamento deste tipo de diabetes consiste na administração subcutânea de insulina exógena. Recentemente, foi demonstrado que o transplante de ilhotas pancreáticas é capaz de tornar o portador de diabetes tipo 1 independente de insulina exógena. Apesar do sucesso alcançado, a necessidade permanente de imunossupressão é uma das principais barreiras para que o transplante de ilhotas possa ser realizado em número maior de pacientes. Assim, o desenvolvimento de novas metodologias que evitem a rejeição do enxerto, como o macro e o microencapsulamento de ilhotas, continua sendo crucial para o estabelecimento definitivo do transplante de ilhotas como opção terapêutica no tratamento de diabetes tipo 1. Neste trabalho, foi padronizado um modelo animal para avaliar, in vivo, a funcionalidade das ilhotas pancreáticas humanas isoladas e purificadas na Unidade de Ilhotas Pancreáticas Humanas do IQUSP. Ratos NIH nude foram tornados diabéticos através de injeção de estreptozotocina para o implante de ilhotas pancreáticas humanas nuas e microencapsuladas. As ilhotas foram microencapsuladas em Biodritina, um novo heteropolissacarídeo patenteado e cedido ao nosso laboratório, tendo sido possível padronizar a produção de microcápsulas uniformes e homogêneas, com tamanho médio entre 400&#181;m e 600 &#181;m. A reversão do diabetes ocorreu em 24% dos ratos nude transplantados com ilhotas pancreáticas humanas nuas. Por outro lado, não observamos reversão do diabetes quando ilhotas encapsuladas foram implantadas, apesar do teste de atividade funcional realizado in vitro ter demonstrado que elas continuam a secretar insulina e a responder ao estímulo com glicose após o encapsulamento. Para elucidar este efeito, cápsulas vazias foram implantadas em ratos nude e em ratos imunocompetentes, os quais desenvolveram processo inflamatório acompanhado de processo fibrótico no local do implante. Estudo imuno-histoquímico está sendo realizado para esclarecer a natureza e a intensidade destes processos. / Type 1 diabetes mellitus results from insufficient or absence of insulin production, as a consequence of destruction of pancreatic &#946; cells, by an auto-imune mechanism. Treatment for this type of diabetes consists of subcutaneous administration of exogenous insulin. Recently, it has been demonstrated that pancreatic islet cell transplantation is capable of rendering type I diabetic patients independent of exogenous insulin. However, in spite of the success achieved, permanent immunosuppression is still required, being the main barrier to expand this treatment to a large number of patients. Therefore, development of new technologies, such as islet macro and microencapsulation to avoid rejection of the tissue implanted, is still crucial for definitive establishment of islet transplantation as a therapeutic alternative for type I diabetes. In the present work, an animal model was established for in vivo evaluation of the functional ability of human pancreatic islets, which were isolated and purified at the Human Pancreatic Islet Unit of the University of São Paulo Chemistry Institute. Diabetes was induced in NIH nude rats through streptozotocin injection followed by implantation of naked or microencapsulated human pancreatic islets. Biodritin, a new and patented heteropolyssaccaride was used to microencapsulate the islets. The production of uniform and homogeneous microcapsules with diameters in the range of 400&#181;m e 600 &#181;m was successfully established. Reversion of diabetes occurred in 24% of the nude rats transplanted with human pancreatic islets. On the other hand, no reversion of diabetes was observed when encapsulated islets were implanted, although their functional activity in vitro indicated that they secreted insulin and responded to glucose stimulation upon encapsulation. In order to elucidate this effect, empty capsules were implanted in nude rat and in immunocompetent rats, both of which developed an inflammatory process accompanied by a fibrotic process in the site of the implant. Immunohistochemical studies are underway to address the nature and the intensity of these inflammatory processes.