Orientador: Mario José Abdalla Saad / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-21T17:03:51Z (GMT). No. of bitstreams: 1 Araujo_TiagoGomes_D.pdf: 9136785 bytes, checksum: 25c68d3dd56714d164986b415f98070d (MD5) Previous issue date: 2012 / Resumo: A resistência à insulina está presente na obesidade e na diabetes tipo 2, e está associada à hiperplasia das ilhotas pancreáticas e hiperinsulinemia como resposta compensatória, entretanto, as forças motrizes por trás desse mecanismo compensatório não são totalmente compreendidos. Dados anteriores sugeriram o envolvimento de um fator circulante desconhecido que na resistência à insulina atua como um fator de crescimento das células ?. Neste contexto, procurando por candidatos a serem este fator circulante, percebemos que o fator de crescimento de hepatócitos (HGF) é um forte candidato a ser este elo entre a resistência à insulina e o aumento da massa de ilhotas / hiperinsulinemia. Nossa abordagem teve como objetivo mostrar uma possível relação de causa-efeito entre o aumento dos níveis circulantes de HGF e a hiperplasia da ilhota / hiperinsulinemia compensatória, assim mostrando a força da associação. Ainda, se esta associação, apresenta ou não uma resposta dose-dependente, temporalidade, consistência, plausibilidade e reversibilidade. Nesse sentido, os nossos dados mostraram: a) uma correlação forte e consistente entre o HGF e o mecanismo de compensação em três modelos animais de resistência à insulina; b) o HGF aumenta a massa de célula ? de uma forma dose-dependente; c) o bloqueio do HGF interrompe os mecanismos de compensação; d) o aumento nos níveis de HGF precede a resposta compensatória associada com a resistência à insulina, indicando que estes eventos ocorrem em um modo sequencial. Além disso, o bloqueio do receptor de HGF (Met) piorou a já prejudicada sinalização da insulina no fígado de ratos obesos induzidos por dieta. Em geral, os nossos dados indicam que o HGF é um fator de crescimento que desempenha um papel fundamental no aumento da massa de ilhotas e hiperinsulinemia em ratos obesos induzidos por dieta, e sugerem um efeito protetor da interação HGF-Met na sinalização de insulina no fígado / Abstract: Insulin resistance is present in obesity and in type 2 diabetes, and is associated with islet cell hyperplasia and hyperinsulinemia but the driving forces behind this compensatory mechanism are incompletely understood. Previous data have suggested the involvement of an unknown circulating insulin resistance-related ?-cell growth-factor. In this context, looking for candidates to be a circulating factor, we realized that hepatocyte growth factor (HGF) is a strong candidate as a link between insulin resistance and increased mass of islets/hyperinsulinemia. Our approach aimed to show a possible cause-effect relationship between increase in circulating HGF levels and compensatory islet hyperplasia/hyperinsulinemia by showing the strength of the association, whether or not is a dose-dependent response, the temporality, consistency, plausibility and reversibility of the association. In this regard, our data showed: a) a strong and consistent correlation between HGF and the compensatory mechanism in three animal models of insulin resistance; b) HGF increases ?-cell mass in a dose-dependent manner; c) blocking HGF shuts down the compensatory mechanisms; d) an increase in HGF levels seems to precede the compensatory response associated with insulin resistance, indicating that these events occur in a sequential mode. Additionally, blockages of HGF receptor (Met) worsen the impaired insulin-induced insulin signaling in liver of diet-induced obesity rats. Overall, our data indicate that HGF is a growth factor playing a key role in islet mass increase and hyperinsulinemia in diet-induced obesity rats, and suggest a protective effect of the HGF-Met axis on insulin signaling in the liver / Doutorado / Medicina Experimental / Doutor em Ciências

O tamanduá-bandeira Myrmecophaga tridactyla é uma espécie considerada “vulnerável” no Brasil, por estar ameaçado de extinção em algumas regiões do país. O presente projeto teve por objetivo identificar e caracterizar as estruturas macro e microscópicas do pâncreas nessa espécie. Para tanto, foram dissecados 16 pâncreas de tamanduás-bandeira provenientes do Hospital Veterinário “Dr. Halim Atique” do Centro Universitário de Rio Preto (UNIRP). As amostras coletadas, foram provenientes de casos de animais atendidos no referido Hospital e que vieram a óbito. O pâncreas situava-se no antímero esquerdo do corpo do animal, apresentava coloração pálida, corpo central e superfície lobulada. Acompanhava a curvatura ventricular maior do estomago aderindo-se na porção inicial do duodeno. Relaciona-se crâniodorsalmente com o baço e ventrículo gástrico, e caudoventralmente com a cápsula fibrosa renal (que aloja o rim esquerdo) e intestinos. Estruturalmente, o órgão demonstrou duas partes distintas: a primeira delas com características exócrinas, composta por ácinos pancreáticos e a segunda endócrina, formada pelas ilhotas pancreáticas encontradas nas regiões media, caudoventral e lobar esquerda. A analise ultraestrutural permitiu identificar nas células centro-acinosas do pâncreas vesículas com grânulos de zimogênio, mitocôndrias, Aparelho de Golgi e retículo endoplasmático rugoso / The giant anteater Myrmecophaga tridactyla is a species considered 'vulnerable' in Brazil since it is threatened in some Brazilian regions. This study aimed to identify and characterize morphological structures of the pancreas in this species. For this, 16 anteaters pancreas from the Veterinary Hospital "Dr. Halim Atique at University Center of Rio Preto (UNIRP), were dissected. All samples were from animals treated at the hospital which died of natural causes. The pancreas was located in the left antimere of the animal’s body, being lobulated and having a pale color and central body. It followed the greater curvature of the stomach, adhering on the initial portion of the duodenum. It was craniodorsally related to the spleen and gizzard, and caudoventrally to the renal fibrous capsule (which houses the left kidney) and intestines. Structurally, the organ had two distinct parts: an exocrine, composed of pancreatic acini; and and endocrine, formed by pancreatic islets found in the medial, caudoventral and left lobar regions. The ultrastructural analysis allowed identifying the central-acinar pancreatic cells with vesicles zymogen granules, mitochondria, Golgi apparatus and rough endoplasmic reticulum

Orientador: Everardo Magalhães Carneiro / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-26T17:22:21Z (GMT). No. of bitstreams: 1 Goncalves_LucianaMateus_M.pdf: 1375240 bytes, checksum: fe3cbb519b479a70d1a3fcd2f6fd3b82 (MD5) Previous issue date: 2015 / Resumo: incidência do diabetes mellitus tipo 2 vem crescendo em todo o mundo, e a estimativa para os próximos anos é que continue aumentando. Dentro desse panorama, destacamos estudos relacionados com substâncias classificadas como desreguladores endócrinos, caracterizados tanto por alterar a síntese, liberação e ação de hormônios bem como atuar sobre receptores hormonais. Dentre os desreguladores endócrinos, alguns apresentam potencial obesogênico e diabetogênico, como é o caso do Bisfenol A (BPA). Estudos demonstram que o BPA, substância utilizada na manufatura do plástico policarbonato e resina epóxi, possui ação sobre receptores de estrógeno. Devido sua ampla aplicação em itens utilizados cotidianamente, estamos constantemente expostos ao BPA desde o desenvolvimento fetal. O objetivo desse estudo foi investigar o efeito da exposição de ilhotas pancreáticas de camundongos neonatos ao BPA. Foram utilizadas ilhotas de neonatos (4 a 6 dias de vida) cultivadas por 48 horas com 0,1nM de BPA para avaliar: 1) secreção de insulina; 2) expressão gênica; 3) conteúdo proteico. Os resultados foram analisados pelo teste t-Student, com nível de significância p<0,05. A secreção de insulina induzida por glicose ou solução de KCl 40 mM foi prejudicada nas ilhotas tratadas com BPA. O conteúdo total de insulina, assim como a expressão dos genes da insulina não foram alterados. Houve redução tanto da expressão gênica como do conteúdo proteico de conexina 36 nas ilhotas tratadas. O BPA reduziu a expressão gênica da subunidade beta 2 do canal de cálcio do tipo L e o conteúdo proteico das subunidades kir6.2 e sur1 do canal de potássio ATP-dependente. O conteúdo de vamp2 e sintaxina 1A também foi menor nas ilhotas tratadas. Concluímos que a exposição das ilhotas de neonatos ao BPA na concentração e tempo utilizados promove alterações dos mecanismos moleculares envolvidos na secreção de insulina / Abstract: Type 2 diabetes is increasing worldwide. Recently, studies have shown the diabetogenic effects of some chemical components used in packages of food and beverages. An obsesogenic and diabetogenic substance is the bisphenol A (BPA), used in manufacture of polycarbonate plastic and epoxy resin. Our exposure to this substance may begin during fetal development and in the first hours of life still in hospital. The aim of this study was to investigate the effects of BPA in islets from newborn mice. Neonate pancreatic islets (4-6 days) were isolate by collagenase method and were cultured with BPA (0.1 nM) for 48 hours, and then used to evaluate: 1) insulin secretion; 2) gene expression; 3) protein content. The results were analyzed by Student t-test, p<0.05. We observed a reduced insulin secretion in response to both glucose and 40 mM KCl solution. However, total insulin content and Ins1/Ins2 gene expression was not altered. On the other hand, connexin 36 gene and protein expression were decreased. Another genes involved with b-cell maturation were unchanged. Furthermore, BPA decreased gene expression of beta 2 subunit of L-type calcium channel. Protein content of K-ATP channel (kir6.2 and sur1), vamp2 and syntaxin 1A were also reduced. We concluded that 0.1 nM BPA exposure for 48 h alters molecular mechanisms involved with insulin secretion from newborn islets in culture / Mestrado / Fisiologia / Mestra em Biologia Funcional e Molecular

Hintergrund/Ziel: Um Langzeitkomplikationen bei Diabetes mellitus Typ 1 (T1D) zu verhindern, können Spenderinseln transplantiert werden, was allerdings lebenslange rigorose Immunsuppression erfordert. Durch Verkapselung der Inselzellen kann die Immunsuppression umgangen werden, aber Upscaling ist schwierig. Ziel dieser Arbeit war es, eine Strategie für das Plotting skalierbarer, semipermeabler, makroporöser Scaffolds mit einem großen Oberfläche-zu-Volumen-Verhältnis zu entwickeln, um lebensfähige und funktionsfähige Pankreasinseln zu Plotten. Methoden: Die für diese Arbeit verwendete Hydrogelmischung bestand aus 3 % Alginat mit 9 % Methylcellulose (MC) als Verdickungsmittel, um eine plottbare Paste herzustellen. Die existierende Mischung musste für die Verwendung von hochaufgereinigtem, d. h. nicht immunogenem ('clinical-grade') Alginat anstelle des zuvor verwendeten zellverträglichen, aber weniger streng aufgereinigten ('research-grade') Alginates adaptiert werden. Für die Charakterisierung der zellfreien Mischung wurden immer Vergleiche zwischen Pasten gezogen, die mit den beiden verschiedenen Alginaten hergestellt wurden. Die Stabilität wurde über rheologische Messungen und die Freisetzung von Ionen und MC bestimmt. Die Permeabilität für Glukose und Insulin wurde mittels Aufnahme- und Freisetzungs-Assays sowie in einem für diese Arbeit entwickelten Diffusionskammersystem analysiert. Da sich Alginatgele als ausreichend permeabel erwiesen haben und eingekapselte Inseln ihre Funktionalität behalten, wurde die Permeabilität von Alginat- und Alg/MC-Gelen verglichen. In früheren Veröffentlichung wurde Alg/MC bereits als kompatibel mit dem Plotting von Einzelzellen beschrieben. Um die Kompatibilität mit endokrinen Zellen zu testen, wurde die β Zelllinie INS-1 verwendet und in Alg/MC Pasten, die mit unterschiedlich sterilisierter MC hergestellt worden waren, getestet. Für pankreatische Inseln, die als Zellcluster empfindlicher auf Scherstress reagieren als Einzelzellen, wurde erfolgreich ein Workflow für das Einbringen in die hochviskose Mischung entwickelt. Dabei wurden die Inseln mit einem Spatel vorsichtig in das Material gefaltet und mit einer Nadel mit einem Innendurchmesser von 840 µm geplottet. Das Plotten pankreatischer Inseln wurde sowohl mit adulten Inseln aus der Ratte als auch mit neonatalen insel-ähnlichen Clustern aus dem Schwein (NICC) durchgeführt und auf Verteilung, Überleben, Apoptose und das Vorhandensein von Hormonen mit Färbungen getestet. Die Funktionalität wurde über Glukose-stimulierter Insulinsekretion (GSIR) analysiert, wobei Inseln Insulin in Reaktion auf hypoglykämische (3,3 mM Glukose) oder hyperglykämische (16,4 mM Glukose) Bedingungen freisetzen, welches im Überstand nachgewiesen wurde. Ergebnisse: Pasten, die mit den verschiedenen Alginaten hergestellt wurden, zeigten eine vergleichbare Viskosität, die für die Herstellung stabiler Strukturen geeignet ist. Clinical-grade Scaffolds hatten eine leicht geringere Vernetzungsdichte, was auf Unterschiede im M:G-Verhältnis zurückgeführt wurde. Mit 70 mM SrCl2 vernetzte Scaffolds blieben in RPMI+ über 21 Tage stabil. Die anhaltende Freisetzung von Vernetzungsionen über den Kultivierungszeitraum war unabhängig vom Alginattyp, aber abhängig von der Art des verwendeten Mediums. Innerhalb der ionisch vernetzten Scaffolds liegt die umgekehrt thermisch gelierende MC bei 37°C mit hoher Wahrscheinlichkeit teilweise geliert vor. Die Freisetzung von nicht gelierter MC konnte in Abhängigkeit der Temperatur gezeigt werden und wurde in allen getesteten Scaffold-varianten in unterschiedlicher Menge beobachtet. Bei Permeabilitätsanalysen folgte die im Kammersystem beobachtete allgemeine Kinetik der Diffusion dem normalen Verlauf der Diffusion durch Hydrogele. Die Permeabilität für Glukose war zwischen den Materialien vergleichbar, d.h. es konnte weder ein Einfluss des Alginat-Typs und der Vernetzungsdichte, noch des Vorhandenseins bzw. der Freisetzung von MC über die Zeit nachgewiesen werden. Die Permeabilität für Insulin muss aufgrund der Bindung an die Diffusionskammer und der mangelnden Stabilität der Gelscheiben in weiteren Experimenten verifiziert werden. Eine vorläufige Schlussfolgerung aus den hier vorgestellten Ergebnissen ist eine leicht verringerte Permeabilität in Alg/MC-Gelen im Vergleich zu reinen Alginatgelen. INS-1 wurden erfolgreich in Alg/MC geplottet. Die Überlebensrate direkt nach dem Plotten war in Anbetracht der Raten immortalisierter mesenchymaler Stammzellen vergleichsweise gering, INS-1 erholten sich jedoch innerhalb einer Woche und proliferierten innerhalb der Scaffolds zu großen metabolisch aktiven Zellclustern. Dies war abhängig von der Sterilisationsmethode: Die Verwendung autoklavierter sowie UV-sterilisierter MC resultierte in Pasten die das Überleben unterstützten. Im Gegensatz dazu führte die Verwendung von mit überkritischem CO2 sterilisierter MC nicht zur Entwicklung von Zellclustern. Metabolisch aktive, Insulin enthaltende Ratteninseln lagen innerhalb der Scaffolds gleichmäßig verteilt vor. Vergleichbar mit Kontrollinseln in Suspensionskultur betrug die Viabilität geplotteter Inseln über einen Zeitraum von 14 Tagen 70-80 %. Der DNA-Gehalt der Scaffolds reduzierte sich über insgesamt 21 Tage stark. In allen über 7 Tage analysierten Inseln war eine begrenzte Menge apoptotischer Kerne vorhanden. In den Kontrollinseln waren diese bevorzugt zentral, in den geplotteten Inseln bevorzugt peripher lokalisiert. Die Anzahl der apoptotischen Kerne unterschied sich zwischen den Kontrollinseln und den geplotteten Inseln nicht signifikant. Insgesamt blieben die Morphologie und die Viabilität der Inseln während des Einbringens in die Mischung und beim Plotten erhalten. Die pankreatischen Hormone Insulin und Glukagon wurden in der Kontrolle und der geplotteten Inseln in angemessener Verteilung und Lokalisation nachgewiesen. Die Stimulation der Ratteninseln wurde über insgesamt 7 Isolationen verifiziert und zeigte eine geringere ab-solute Insulinsekretion aus den geplotteten Inseln als aus den Kontrollinseln, aber an Tag 4 und 7 der Kultur einen vergleichbaren aus dem GSIR berechneten Stimulationsindex (SI). Darüber hinaus konnte gezeigt werden, dass die Insulinsekretion bei sukzessiv variierender Stimulation mit Glukose dem Verlauf der Glukosekonzentration folgt. Dies zeigt, dass die geplotteten Inseln die externe Glukosekonzentration nachweislich wahrnehmen und entsprechend reagieren. Zusammenfassend lässt sich festhalten, dass die absolute Insulinsekretion aus den geplotteten Inseln zwar geringer war als die der Kontrollinseln, die relative Funktionalität in den geplotteten Scaffolds jedoch erhalten blieb. Zur Validierung der Ergebnisse von adulten Ratteninseln wurden in einer vorläufigen Studie die potentiell klinisch translatierbaren, aber empfindlicheren, NICC verwendet. Metabolisch aktive, Insulin enthaltende NICC, lagen innerhalb der Scaffolds gleichmäßig verteilt vor. Die Viabilität der NICC war mit etwa 60 % vergleichsweise gering, aber vergleichbar mit der der Kontroll-NICC und änderte sich über einen Kultivierungszeitraum von 21 Tagen nicht signifikant. In allen geplotteten NICC konnten lebendige Zellen nachgewiesen werden, während eine geringe Anzahl Kontroll-NICC in der Lebendfärbung fast kein Signal zeigten. Die Anzahl der apoptotischen Kerne nahm im Verlauf von 7 Tagen bei den geplotteten, nicht aber bei den Kontroll-NICC signifikant zu. Die pankreatischen Hormone Insulin, Glukagon und Somatostatin wurden in einer geringen Anzahl von zufällig innerhalb der Cluster verteilten Zellen über einen Zeitraum von 7 Tagen nachgewiesen. Die Funktionalität wurde weder durch das Einbringen in das Material noch durch das Plotten beeinflusst. Unter Verwendung eines GLP-1-Analogons zur Amplifikation der Reaktion zeigten geplottete und Kontroll-NICC eine vergleichbare Funktionalität. Der SI nahm im Laufe der Kultivierungszeit jedoch ab und lag nach 14 bzw. 21 Tagen Kultur in geplotteten und Kontrollproben unter 2. Schlussfolgerungen: Die adaptierte Alg/MC-Mischung zeigte ausreichende Stabilität und Permeabilität für das Plotten pankreatischer Inseln. In einer Proof-of-Concept-Studie mit adulten Ratteninseln wurde gezeigt, dass die hier adaptierte Alg/MC-Mischung generell für das Plotting lebendiger und funktionaler pankreatischer Inseln geeignet ist. Vorläufige Ergebnisse, die mit einer geringen Anzahl von Wiederholungen und Proben erstellt wurden, deuten darauf hin, dass Alg/MC auch ein grundlegend geeignetes Material für das Plotten von NICC ist. Die weitere Charakterisierung und insbesondere die weitere Materialadaption zur Unterstützung des Überlebens und der Ausreifung von NICC in vitro werden in einer Folgestudie vorgenommen werden.:Table of contents Abbreviations 1 1 Motivation 3 2 Introduction and state of the art 5 2.1 Pancreatic islets and diabetes mellitus type 1 5 2.1.1 Anatomy and function of the pancreas and pancreatic islets 5 2.1.2 Insulin biosynthesis, release and function 7 2.1.3 Diabetes mellitus type 1 (T1D) 9 2.1.4 Current treatment options for T1D 11 2.2 Surgical replacement of β-cells 13 2.2.1 Islet transplantation 13 2.2.2 Xenotransplantation 15 2.2.3 Encapsulation of islets 17 2.3 3D bioprinting in medical research 22 2.3.1 Extrusion-based 3D bioprinting 22 2.3.2 Bioplotting of islets 24 3 Materials & Methods 26 3.1 Cell culture 26 3.1.1 Cell lines 26 3.1.2 Primary islets from rat 26 3.1.3 Neonatal porcine islet-like cell clusters 27 3.2 Material preparation and characterisation 27 3.2.1 Hydrogel preparation 27 3.2.2 3D plotting of cell-free hydrogels for material characterisation 28 3.2.3 Rheological characterisation 28 3.2.4 Quantification of ion release 29 3.2.5 Determination of methylcellulose content 29 3.2.6 Preparation of cell-free hydrogel discs 29 3.2.7 Permeability measurements 30 3.3 Plotting and characterisation of cell-laden constructs 32 3.3.1 Incorporation of cells & scaffold preparation 32 3.3.2 Staining methods for the characterisation of (embedded) cells 32 3.3.3 Functional analysis of islets: Glucose stimulated insulin release (GSIR) 34 3.4 Statistics 35 4 Results 36 4.1 Adaptation & characterisation of cell-free Alg/MC 36 4.1.1 Paste viscosity and scaffold stability 36 4.1.2 Scaffold composition during incubation under cell culture conditions 38 4.1.3 Permeability for glucose & insulin 42 4.2 Adaptation & characterisation of islet cell incorporation into the Alg/MC blend 58 4.2.1 Sterilisation of MC: influence on β-cell survival and behaviour 58 4.2.2 Incorporation of pancreatic islets into the highly viscous Alg/MC blend 61 4.2.3 Needle diameter for the plotting of pancreatic islets 64 4.3 Plotting of adult murine pancreatic islets 66 4.3.1 Distribution, morphology and viability of bioplotted murine islets 66 4.3.2 Functionality of bioplotted murine islets 74 4.4 Plotting of neonatal porcine islet-like cell clusters (NICC) 80 4.4.1 Distribution, morphology and viability of bioplotted NICC 80 4.4.2 Functionality of bioplotted NICC 87 5 Discussion 92 5.1 Cell-free Alg/MC 93 5.1.1 Gel viscosity and crosslinking of alginate 93 5.1.2 Release of MC 99 5.1.3 Permeability for glucose & insulin 106 5.2 Cell incorporation into Alg/MC 118 5.2.1 Incorporation of β-cells 118 5.2.2 Incorporation of pancreatic islets 119 5.3 Plotting of adult murine pancreatic islets 121 5.3.1 Distribution, morphology and viability of bioplotted murine islets 121 5.3.2 Functionality of bioplotted murine islets 124 5.4 Plotting of neonatal porcine islet-like cell clusters (NICC) 127 5.4.1 Distribution, morphology and viability of bioplotted murine islets 127 5.4.2 Functionality of bioplotted NICC 130 Summary 134 Zusammenfassung 137 References 140 Addendum 172 A.1 Supplementary data for the results 172 Supplementary data for “4.1.1. Paste viscosity and scaffold stability” 172 Supplementary data for “4.1.2 Scaffold composition during incubation under cell culture conditions” 173 Supplementary data for “4.1.3 Permeability for glucose & insulin” 175 Supplementary data for “4.2.1 Sterilisation of MC: influence on β-cell survival and beha-viour” 178 Supplementary data for “4.3.2 Functionality of bioplotted murine islets” 182 Supplementary data for “4.4.2 Functionality of bioplotted NICC” 187 A.2 Supplementary data for the discussion 189 Supplementary data for “5.1.1. Gel viscosity and crosslinking of alginate” 189 Supplementary data for “5.1.2. MC release” 191 Supplementary data for “5.1.3 Permeability for glucose & insulin” 195 Supplementary data for “5.2.1 Incorporation of β-cells” 197 Supplementary data for “5.4.1 Plotting of neonatal porcine islet-like cell clusters (NICC)” 198 List of figures 196 List of tables 197 List of publications 198 Acknowledgements 200

京都大学 / 新制・課程博士 / 博士(医学) / 甲第23368号 / 医博第4737号 / 新制||医||1051(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 川口 義弥, 教授 妹尾 浩, 教授 稲垣 暢也 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

The purpose of these studies was to investigate the impact of high-fat diet (HFD) exposure during pregnancy and the early post-natal period on fetal and post-natal development of the endocrine pancreas of the Japanese macaque. Specifically I hypothesized that the HFD would alter islet morphology and lead to disturbances in glucose homeostasis in these animals. Adult female Japanese macaques were placed on either a control (CTR) or HFD diet for 4 years. Fetuses were collected at gestational day 130 (G130), while other offspring from the CTR and HFD mothers were carried to term. After birth, infant animals were maintained with their mothers on the same diet then weaned onto either the CTR or HFD diet for five months. Animals were studied up to 13 months of age, yielding 4 postnatal groups: CTR/CTR, CTR/HFD, HFD/CTR and HFD/HFD. Pancreata were collected from these offspring for gene expression and immunohistochemical analysis. Physiological measurements, including body weight, body fat percentage, fasting glucose, insulin, glucagon and response to intravenous glucose tolerance tests (IVGTTs) and an intravenous insulin tolerance test (IVITT) were collected from the post-natal offspring. Total fetal islet mass and β cell mass were not changed, but α cell mass was significantly decreased in HFD fetuses, leading to a significant increase in the β cell to α cell ratio in HFD fetal offspring. The HFD offspring displayed a significant change from CTR offspring in expression of genes involved in glucose homeostasis and islet neogenesis, including PDX1, NeuroD, Glucokinase and Glut2. Postnatal HFD animals were significantly heavier than CTR offspring and had increased adiposity by 6-7 months of age. There was no significant effect on fasting or stimulated insulin secretion at this time point, but HFD offspring were significantly insulin resistant just prior to weaning. At 13 months of age, basal and glucose-stimulated insulin secretion were elevated in HFD/HFD animals and the CTR/HFD group displayed moderate insulin resistance. There was also a significant sex effect, with males from the HFD/CTR and HFD/HFD group having increased body weight and elevated fasting glucose. Although pancreata from both the HFD/HFD and CTR/HFD animals displayed significant changes in expression of genes involved in glucose homeostasis, the pattern was distinct for the two groups. Islet mass was also elevated in both of these groups; yet, HFD/HFD only displayed an increase in β cell area, while CTR/HFD had a concomitant increase in α cell area, which served to normalize the β cell to α cell ratio to control levels. In contrast, the HFD/HFD group exhibited a 40% increase in the β cell to α cell ratio. These studies demonstrate that in-utero exposure to a HFD leads to decreased α cell plasticity in response to chronic post-natal HFD consumption. Animals exposed to the HFD during pregnancy and the early post-natal period become insulin resistant, but remain normoglycemic. HFD consumption during the post-weaning period causes similar complications in glucose homeostasis and islet mass in both the CTR/HFD and HFD/HFD animals. However, there are distinct differences in the molecular and cellular adaptive response between these two groups.

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This study was designed to examine the effect of genistein, a botanical derived primarily from legumes, on pancreatic β-cell proliferation and the related molecular mechanisms. Diabetes mellitus is a major and growing public health problem worldwide. Both in type 1 (T1D) and type 2 diabetes (T2D), the deterioration of glycemic control over time is primarily caused by an inadequate mass and progressive dysfunction of β-cells. Therefore, the search for novel, safe and cost-effective agents that can enhance islet β-cell proliferation, thereby preserving β-cell mass, could be one of the essential strategies to prevent diabetes, given that β-cells have the potential to regenerate by proliferation of pre-existing b-cells in both physiological condition and after onset of diabetes. Genistein has various biological actions. However, studies on whether genistein has an effect on pancreatic β-cell function are very limited. Our laboratory recently found that genistein activates cAMP/protein kinase A (PKA) signaling in both clonal β-cells and mouse islets. Here I present evidence that genistein induced cellular proliferation of clonal rat pancreatic β-cells (INS1) and human islets following 24 h of incubation. This effect was dose-dependent with 5 µM genistein inducing a maximal 41% increase. The effect of genistein on cell proliferation was not dependent on estrogen receptors because this effect was not blocked by the estrogen receptor inhibitor ICI182,780. In addition, the genistein effect on β-cell proliferation was not shared by 17-β-estradiol or a host of structurally related flavonoid compounds, suggesting that this genistein action is structure-specific. Pharmacological or molecular intervention of PKA or MEK1/2, the upstream kinase of p42/44 mitogen activated protein kinases (ERK1/2), completely abolished the genistein-stimulated proliferation of INS1 cells and human islets, suggesting that both molecules are essential for genistein action. Consistent with its effect on cell proliferation, genistein increased intracellular cAMP and subsequently activated PKA in human islets. Genistein also caused a rapid and sustained phosphorylation of ERK1/2 with a maximal increase of 185% at 5 µM genistein. The genistein-induced ERK1/2 activation was completely ablated by inhibition of PKA in INS1 cells and human islets. Furthermore, I found that genistein induced protein expression of cyclin D1, a nuclear target of PKA and ERK1/2 activation and a major cell-cycle regulator essential for ï ¢-cell growth. These findings demonstrated that genistein may be a plant-derived growth factor for pancreatic β-cells involving induction of cyclin D1 via activation of the cAMP/PKA-dependent ERK1/2 signaling pathway, thereby providing a novel role for genistein in the regulation of pancreatic β-cell function. / Master of Science