Thesis--University of Adelaide, 1957. / Typewritten.

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This document only includes an excerpt of the corresponding thesis or dissertation. To request a digital scan of the full text, please contact the Ruth Lilly Medical Library's Interlibrary Loan Department (rlmlill@iu.edu). / This investigation was initiated to test the hypothesis that streptozotocin (STZ) and N-methylnitrosourea (MNU) damage B-cells of islet of Langerhans by depleting islets pyridine nucleotide content. Islets of Langerhans isolated by the collagenase method from nonnal rats were exposed to STZ rmder various experimental conditions and islets pyridine nucleotide content was temporally correlated with cytotoxicity. Metabolic and insulin secretory function of islets of Langerhans were used as indices of B-cell toxicity. The nitrosoureas (STZ and MNU) exerted time-, dose-, and temperature-dependent suppression of glucose-stimulated insulin secretion. Identical studies indicated that rmlike the nitrosoureas, alloxan was B-cytotoxic at 0°c even in the presence of 16.7 mM glucose. Whereas 2 mM alloxan elicited transient but consistent release of insulin by islets perifused in low glucose (1.7 mM) at 37°c, there was no release of insulin by islets perifused with 5 mM STZ, The effect of 5 mM STZ or of 10 mM MNU can be completely inhibited by the simultaneous presence of nicotinamide, isonicotinamide, picolinamide and other primary amides (e.g. pyrazinamide and benzamide) at concentration of 20 mM. Glucose (16.7 mM), pyridine nucleotides (10 mM) and acid derivative of amides (e.g. nicotinic acid; 20 mM) were ineffective. 2-Deoxyglucose (20 mM) or 3-0-methylglucose (20 mM) offered partial protection. After exposure of islets to STZ (5 mM) or MNU (10 mM), complete reversal of the effect was obtained with nicotinamide (20 mM) or its isomers, with time of exposure not exceeding 30 minutes. Beyond 30 minutes, the reversibility was partial or absent, except in the presence of 0.5 mM phenazine methosulfate (PMS) which induced release of insulin in both normal and nitrosoureas pre-treated islets. Insulin releasing action of PMS was dose-, time- and temperature-related; occurred even in the absence of glucose; was inhihitcd hy epinephrine (10 mM, hut not by mannoheptulose (20 mM); and was not potentiatcd by cyclic AMP (5 mM) or theophylline (10 mM). In the perifusion system, the patterns of response induced by PMS (0,5 mM) was spike-like release reaching a maximum in 5 minutes and declining rapidly to half-maximal value in 10 minutes. Normal islets pre-exposed to PMS was refractory to subsequent glucose (16.7 mM) stimulation. Islets pre-treated with STZ (1-5 mM) metaholized less 14c-glucose than control islets. The order of inhibition hy STZ of 14c-glucose metabolism by islet was: l-14C->U- 14C->6-14C-glucose. PMS (0.5 mM) augmented the metabolism of U-14C- and 1-14C glucose by STZ pre-treated islets. However, the metabolism of 6-14C-glucose was unelevated by PMS. The level of NADP+ + NADPII but not the level of NAD, decreased after two minutes exposure of islets to STZ. At thirty minutes, however, the levels of NAD,6-phosphogluconate and NADP+ + NADPII were decreased. The depletion paralleled suppression of insulin secretion. The level of NADP+ + NADPH in islets was decreased more than the level of NAD. Whereas PMS (0.5 mM) elevated the level of NADP+ + NADPH, the level of NAD was not augmented. Islets isolated from rats 3 or more hours after pre-injection with STZ (65 mg/kg) or 6-aminonicotinamide (40 mg/kg) failed to secrete insulin in response to glucose stimulation. However, insulin secretion by such islets was elevated in the presence of PMS (0.5 mM). Whereas PMS induced insulin secretion was much reduced 48 hours after preinjection of rats with STZ, the secretory activity in islets of rats 48 hours after 6-aminonicotinamide injection was slightly decreased. Pyridine nucleotides augmented secretion only in islets of 6-aminonicotinamide pre-injected rats. It is concluded that the immediate response of islets to nitrosoureas in vitro differs from islets response to alloxan. The actions of STZ and MNU are qualitatively similar. The B-cytotoxic effect of nitrosoureas is exerted directly or indirectly on the metabolic and energy coupling functions of pyridine coenzymes. Such an effect could be reversed by supplying the islets with reactive proton donors as substitutes for pyridine nucleotides.

Thesis (Ph. D.)--University of Wisconsin--Madison, 1957. / Typescript. Abstracted in dissertation abstracts, v. 16 (1956) no. 11, p. 2020-2021. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves 169-175).

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Pacientes diabéticos apresentam maior severidade e prevalência de doenças periodontais. Em ratos diabéticos, a doença periodontal induzida com ligaduras e inoculação de bactérias é mais severa. Contudo, na ausência de estímulos agressivos como ligaduras, a influência do diabetes sobre o periodonto de rato é pouco conhecida. O objetivo deste estudo foi avaliar a presença e a progressão da doença periodontal por longos períodos apenas com a indução do diabetes. O diabetes foi induzido em ratos Wistar (n=25) pela administração endovenosa de 42mg/kg de aloxana e, juntamente com os animais controle (n=25), foram analisados nos períodos de 1, 3, 6, 9 e 12 meses. Os animais foram sacrificados tendo suas hemimandíbulas removidas, radiografadas e submetidas aos procedimentos histotécnicos. A mensuração da altura da crista óssea e dos valores de pixel foi realizada pelo programa ImageJ em radiografias previamente escaneadas. A altura da crista óssea nos animais diabéticos foi significativamente menor em relação ao grupo controle nos tempos de 3, 6, 9 e 12 meses (p<0,05 ANOVA). Contudo, não foram observadas diferenças na média dos valores de pixel entre os grupos controle e diabético em tempos correspondentes (p<0,05 ANOVA). A análise histológica apresentou redução na densidade em volume da fibras colágenas, fibroblastos e vasos sangüíneos. Observou-se aumento dos osteoclastos e células inflamatórias (p<0,05 ANOVA). Além disso, cáries rampantes foram detectadas no grupo diabético. Os resultados sugerem que o diabetes sem reposição hormonal, mesmo na ausência de fatores locais, promove alterações teciduais características do desenvolvimento da doença periodontal em ratos. Assim, o diabetes pode ser considerado como agente responsável pela indução, progressão e manutenção da doença periodontal em ratos. / Periodontal disease in diabetic patients present higher severity and prevalence, and increased severity of ligature-induced periodontal disease is verifyed in diabetic rats. However, in the absence of aggressive stimuli such as ligatures, the influence of diabetes over periodontal tissues of rat is unknown. The aim of this study was evaluate the stablishment and progression of periodontal diseases in rats only with the induction of diabetes. Diabetes was induced in Wistar rats (n=25) by endovenous administration of 42mg/kg of alloxan, and together with control animals (n=25), were analyzed at 1, 3, 6, 9 and 12 months after diabetes induction. The animals were sacrificed and the jaws removed, radiographed and submitted to the histopathological procedures. The measurement of alveolar bone crest height and pixel values was carried through the ImageJ program in scanned radiographs. It was observed a reduction in the height of the bone crest in the diabetic animals at the times of 3, 6, 9 and 12 months (p<0,O5 ANOVA). However, no differences in the average of pixel values between control and diabetic groups were observed (p<0,O5 ANOVA). The histopathological analyses of the diabetics animals showed reduction in collagen fibers, fibroblasts and blood vessels. In addition, it was observed an increase in in the number of osteoclasts and inflammatory cells (p<0,O5 ANOVA). Moreover, severe caries were detected in the diabetic group.The results suggest that diabetes without hormonal reposition, and in the absence of aggressive factors as ligatures, result in tissular alterations typical of periodontal diseases. In this way, diabetes can be considered the agent responsible for the induction, maintenance and progression of the periodontal disease.

Indivíduos diabéticos apresentam incidência elevada de doenças infecciosas. Isto pode estar relacionado às alterações na capacidade da resposta destes indivíduos aos agentes agressores. Em animais diabéticos, algumas destas alterações já foram descritas, assim como sua reversão pela administração de insulina. Este hormônio regula o metabolismo celular, modulando a atividade de proteínas e mediadores inflamatórios envolvidos neste processo. Sabemos que o lipopolissacarídeo (LPS) estimula, em macrófagos alveolares (MA) de animais não-diabéticos, a liberação do fator de necrose tumoral (TNF)-&#945; e do óxido nítrico (NO). O pré-tratamento deste MA com insulina inibiu todos estes efeitos. Assim, neste projeto, avaliamos o papel da insulina em MA e macrófagos peritoneais (MP) de camundongos, tornados diabéticos pela indução com aloxana (60 mg/kg, i.v.). Uma suspensão contendo 1x106 células foi estimulada com LPS (100 ng/mL) na presença ou não de insulina (1mU/mL). Realizamos a evolução temporal (0,5; 1; 3; 6; 24 horas) para a dosagem de NO, TNF-&#945; e interleucina (IL)-10. Nos tempos de maior produção destas citocinas (0,5 e 3 horas), também quantificamos IL-6, interferon (IFN)-&#947; e IL-4. Nossos resultados mostram que a produção/liberação dos mediadores imunes por MA e MP estimulados por LPS, quando tratados simultaneamente com insulina, tiveram uma redução. Assim, a produção/liberação de NO foi reduzida durante 0,5; 1; 3; 6, 24 horas, em MA e em MP; a liberação de TNF-&#945; foi reduzida durante 0,5 hora, em MP; a liberação de IL-6 foi reduzida durante 3 horas, em MA, e 0,5 hora, em MP. Estes dados mostram que a insulina reduziu a liberação destes mediadores inflamatórios, tanto para os MA quanto para os MP, durante o estímulo com LPS, de animais diabéticos já nos primeiros minutos de estímulo com LPS, com um pico de redução, na maioria das vezes, em 3 horas. / Diabetic patients exhibit high incidence of infectious diseases, at least in part, by due impaired immune response against aggressive agents. Lipopolysaccharide (LPS) triggers the releasing of tumor necrosis factor (TNF)-alpha and nitric oxide (NO) by alveolar macrophages (AM) of non-diabetic animals. The pretreatment using insulin inhibited of these cytokine release. The aim in the present study was to evaluate the insulin role on releasing of cytokines by MA and peritoneal macrophages (MP) of diabetic mouse. Resident AM and MP from diabetic (alloxan 60 mg/kg, i.v.) male C57BL/6 mice (CEUA/FCF/USP-339) stimulated by LPS (100 ng/mL). Insulin (1 mU/mL) insulin treatment was simultaneously with stimulus by LPS. Cytokines were measured by ELISA, and NO by Griess reaction. We performed a time course (0,5; 1; 3; 6; 24 hours), and measured NO, TNF-alpha and interleukin (IL)-10. We also measured IL-6, interferon (IFN)-&#947;, and IL-4 in 0.5 and 3 hours. Results were evaluated by analysis of variance (ANOVA) followed by multiple comparison Tukey-Kramer or the nonparametric Kruskal-Wallis test. P value were considered when <0.05. Before the induction of diabetes by alloxan injection in day 0 animals showed: weight (26±0,3 g) and blood glucose concentration (164±2,6 mg/dL) - and 10 days after the onset of the disease, diabetic mice presented a lower weight gain (24 ± 0,4 g*) and elevated blood glucose concentration (546±9,7 mg/dL*). An increase was seen NO release levels and TNF-alpha in the time course (Figure 1); an increase of IL-6 in 3 hours (Figure 3), and a decrease of IL-4 in 3 hours (Figure 5) after LPS stimulation of MA in diabetic animals. In MP, there was an increase of NO levels, and TNF-alpha in the time course (Figure 2); in 3 hours increased IL-6 levels (Figure 4) - under the same conditions. Insulin treatment under stimulation by LPS in MA reduced the levels of NO, IL-6, and TNF-alpha compared to the group stimulated by LPS. Regarding MP, insulin treatment also decreased NO levels, TNF-alpha, and IL-6. IL-4 levels produced by MP, and IFN-&#947; levels were not be detected in MA and MP under stimulation by LPS, or in the presence of hormone. These findings suggest that insulin reduce the release of these inflammatory mediators by both resident macrophages of diabetic animals concomitantly stimulation by LPS

Alloxan (ALX) ist ein klassisches Diabetogen, welches in Nagetieren spezifisch pankreatische ß-Zellen zerstört und Symptome induziert, die dem humanen Typ-1-Diabetes vergleichbar sind. Durch eine einmalige, intravenöse (iv) Injektion einer subtoxischen Dosis von 50 mg ALX/kg Körpergewicht (KG) werden Schäden an der ß-Zelle hervorgerufen, die innerhalb von 48-72 Stunden in &#61603;50% der Mäuse einen Diabetes auslösen (Schwellenwert Euglykämie zu Hyperglykämie ist 11,1 mmol/l). Das toxische Potential von ALX besteht in der Generierung von reaktiven Sauerstoffspezies (ROS), vorwiegend Superoxidanion-, Wasserstoffperoxid- und Hydroxylradikalen. Ziel der vorliegenden Arbeit war zu untersuchen, ob durch ALX präferentiell Strukturen der ß-Zelle zerstört werden, die essentiell für die ß-Zellfunktion &#150; die Insulinproduktion &#150; sind. Diese sind u.a. der Glucosetransporter 2 (GLUT2), die Glucokinase und das Proinsulin. Daran anschliessend stellte sich die Frage, ob ALX-Toxizität durch Vorbehandlung mit D-Glucose (D-G), 5-Thio-D-Glucose (5-T-G) oder mit Zink-angereichertem Trinkwasser &#150; zur Anreicherung des Antioxidants Metallothionein - verhindert werden kann. Hierzu wurden männliche C57BL/6- und 129S3-Mäuse entweder einmalig iv mit D-G oder 5-T-G vorbehandelt oder die Mäuse erhielten eine Woche vor der ALX-Injektion und über die gesamte Versuchsdauer hinweg Zink-angereichertes Trinkwasser. Anschliessend wurde der Einfluss auf den ALX-induzierten Diabetes, die orale Glucosetoleranz und die mRNA-Expression der oben genannten Gene mittels RT-PCR untersucht. Der Gesamtinsulingehalt der ALX-behandelten Pancreata wurde über die Bestimmung des immunreaktiven Insulins ermittelt. In der vorliegenden Arbeit wird gezeigt, dass 1.) die Vorbehandlung mit D-G den ALX-induzierten Diabetes signifikant (p&#61603;0,001) verhinderte; 2.) trotz Euglykämie in mit D-G- und ALX-behandelten Mäusen eine pathologische orale Glucosetoleranz über Wochen als ALX-Folgeschaden persistierte; 3.) die Vorbehandlung mit 5-T-G, dem chemisch der D-G ähnlichsten Analog, jedoch den ALX-induzierten Diabetes signifikant (p&#61603;0,001) potenzierte; 4.) Zink-angereichertes Trinkwasser die ALX-induzierte Hyperglykämie signifikant (p&#61603;0,001) reduzierte; 5.) ALX zunächst die mRNA-Expression des GLUT2 signifikant (p&#61603;0,001) reduzierte und nachfolgend auch signifikant (p&#61603;0,05) die mRNA-Expression der Glucokinase, wenn auch weniger ausgeprägt als für die GLUT2-Expression; 6.) ALX keine Veränderung der mRNA-Expression von Proinsulin auslöste; 7.) die Vorbehandlung mit D-G signifikant (p&#61603;0,05) die ALX-induzierte Reduktion der mRNA-Expression von GLUT2 und der Glucokinase verhinderte und 8.) der Insulingehalt im gesamten Pankreas 24 h nach ALX-Injektion signifikant (p&#61603;0,05) reduziert wurde. Es wird geschlussfolgert, dass der GLUT2 und die Glucokinase primäre Zielstrukturen der ALX-Toxizität unter den verwendeten Versuchsbedingungen sind. Diese Läsionen sind die Ursache für den Diabetes. Durch Vorbehandlung mit D-G können der GLUT2 und die Glucokinase vor ALX-Toxizität geschützt werden, obwohl trotz Euglykämie unter physiologischen Bedingungen eine pathologische orale Glucosetoleranz als ALX-Folgeschaden in den Mäusen persistierte. Es muß noch geklärt werden, worin die Gründe für den protektiven Effekt der D-G und den potenzierenden Effekt der 5-T-G liegen und inwieweit ALX-induzierte Radikale selektiv wirksam sind, oder ob diese Selektivität auf anderen Mechanismen, wie z.B. Transkriptionsfaktoren, beruht. Letztendlich zeigen diese Befunde, dass sich der pathogenetische Mechanismus von ALX von anderen Diabetogenen unterscheidet, wie z.B. Streptozotozin, welches selektiv den GLUT2 schädigt, der durch Vorbehandlung mit 5-T-G vor der Streptozotocin-Toxizität geschützt werden kann. Daraus ist abzuleiten, dass es in der Präventivmedizin unterschiedlicher Vorsorgemassnahmen bedarf, um Risiko-patienten vor der Manifestation eines Diabetes mellitus zu schützen. / Type 1 diabetes results from irreversible damage of insulin-producing ß-cells. In laboratory animals, diabetes can be induced with alloxan (ALX). ALX is a potent generator of reactive oxygen species (ROS), which can mediate ß-cell toxicity. However, the initial lesions on essential ß-cell structures are not known. In this study, we analyzed the effect of ALX on the glucose transporter 2 (GLUT2), glucokinase and proinsulin. For this purpose, we investigated the effect of pretreatment with the glucose analogues D-glucose (D-G) and 5-thio-D-glucose (5-T-G), as well as with zinc-enriched drinking water to induce the antioxidant metallothionein, on ALX-induced diabetes, on oral glucose tolerance (OGT) and on the mRNA-expression of the above mentioned genes with semiquantitative RT-PCR in male C57BL/6 and 129S3 mice. The total insulin content of ALX-treated pancreata was determined as immune reactive insulin. One single intravenous (iv) injection of 50 mg ALX/kg body weight (bwt) induced diabetes in &#61603;50% of mice of both strains (blood glucose level &#61603;11.1 mmol/l). One single iv preinjection of D-G prevented significantly (p&#61603;0,001) diabetes in both strains, yet, in these euglycemic mice, an impaired oral glucose tolerance persisted. In contrast, the pretreatment with a single injection of 5-T-G potentiated significantly (p&#61603;0,001) the toxicity of ALX. Administration of zinc-enriched drinking water, however, reduced ALX-induced hyperglycemia (p&#61603;0,001). The mRNA-expression of GLUT2 and glucokinase was time-dependently reduced and the effect was more pronounced for GLUT2 (p&#61603;0,001) than for glucokinase (p&#61603;0,05). The pretreatment with D-G protected against the mRNA-reduction of both GLUT2 and glucokinase (p&#61603;0,05). Interestingly, the mRNA-expression of proinsulin remained unaffected as well as the pancreatic total insulin content. A significant (p&#61603;0,05) reduction of pancreatic insulin content was found after 24 h. In conclusion, ALX exerts differential toxicity on essential ß-cell structures. This toxic effect was more pronounced for GLUT2 than for glucokinase mRNA. Pretreatment with D-G prevented ALX-toxicity, whereas in euglycemic mice an impaired oral glucose tolerance persisted. It has to be elucidated, whether ALX-induced ROS select essential ß-cell structures or whether, as one possibility, transcription factors in the ß-cell are specifically directing ROS to GLUT2 and glucokinase mRNA. Finally, these results differ from those obtained with other diabetogens, e.g., streptozotocin, which exerts selective toxicity on the GLUT2 and which is prevented by 5-T-G. However, diabetogens damage ß-cell function through different pathogenic pathways and, therefore, different interventional regimen may be required to prevent type 1 diabetes in individuals at risk.

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