Thesis (Ph. D.)--University of Lund, 1998. / Originally issued as the author's doctoral thesis. Includes bibliographical references.

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Thèse de doctorat : Biologie cellulaire et moléculaire : Paris 12 : 2007. / Thèse uniquement consultable au sein de l'Université Paris 12 (Intranet). Titre provenant de l'écran-titre. Bibliogr. 350 réf.

Ph.D.

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Dissertação (mestrado)—Universidade de Brasília, Faculdade de Medicina, 2008. / Submitted by Jaqueline Oliveira (jaqueoliveiram@gmail.com) on 2008-12-08T16:24:34Z No. of bitstreams: 1 DISSERTACAO_2008_JoicyFerreiraQueiroz.pdf: 1276087 bytes, checksum: bc45206a6aba69d396a9bc55d1d07410 (MD5) / Approved for entry into archive by Georgia Fernandes(georgia@bce.unb.br) on 2009-02-16T15:27:12Z (GMT) No. of bitstreams: 1 DISSERTACAO_2008_JoicyFerreiraQueiroz.pdf: 1276087 bytes, checksum: bc45206a6aba69d396a9bc55d1d07410 (MD5) / Made available in DSpace on 2009-02-16T15:27:12Z (GMT). No. of bitstreams: 1 DISSERTACAO_2008_JoicyFerreiraQueiroz.pdf: 1276087 bytes, checksum: bc45206a6aba69d396a9bc55d1d07410 (MD5) / A literatura tem mostrado que mamíferos alimentados com dietas ricas em proteína são, em geral, mais resistentes ao jejum. Entretanto, o morcego hematófago Desmodus rotundus, apesar de possuir uma dieta rica em proteína (sangue) é marcadamente susceptível à privação alimentar, apresentando hipoglicemia severa após 24h de jejum. lém disso, esta espécie possui pequenas reservas de glicogênio e lipídios, baixas oncentrações de insulina plasmática e pancreática e um menor número de células á e â em uas Ilhotas de Langerhans. Este trabalho teve como objetivos verificar em D. rotundus: a resposta ao Teste de Tolerância à Glicose intra-peritoneal, (ipGTT); a resposta ao Teste de Tolerância à Insulina intra-peritoneal (ipITT), a secreção de insulina in vitro estimulada por diversos secretagogos, a via tecidual de sinalização da insulina e as concentrações plasmáticas de insulina, glucagon e cortisol em animais alimentados e jejuados. Os resultados do ipGTT mostraram que esses morcegos não conseguem lidar com uma sobrecarga de glicose, sugerindo intolerância à esse substrato. Os experimentos de ipITT mostraram que não houve alteração da glicemia após injeção de insulina, sugerindo que, além de baixas concentrações de insulina, D. rotundus apresenta resistência periférica a este hormônio. As concentrações de insulina e glucagon plasmáticos verificadas em D. rotundus alimentados e jejuados por 24h foram inferiores ao observado para a maioria dos mamíferos estudados até o momento (Bruttomesso et al. 1999; Lacroix et al., 2004; Lephart et al., 2004), inclusive humanos (Corrêa et al., 2007). Por outro lado, foram verificados altos níveis de cortisol plasmático nesta espécie, sugerindo que este hormônio não deve participar de forma importante na manutenção da homeostase glicêmica, pelo menos no jejum, como ocorre normalmente em mamíferos (Rafacho et al., 2007, 2008). O estudo da secreção estática de insulina por fragmentos de pâncreas estimulados por seus principais secretagogos mostrou que as células â de D. rotundus apresentam aumento na taxa de secreção de insulina em resposta a glicose e leucina, mas não reagem à adição de K+ no meio de incubação. Finalmente, não houve alteração nos níveis de fosforilação da Akt, proteína da via de sinalização tecidual da insulina, em resposta à estimulação por esse hormônio no fígado. Já a ERK, proteína da via MAPK da estimulação tecidual da insulina, responde normalmente a este hormônio. Embora tenha havido aumento da expressão tecidual da ERK em resposta à insulina, esta via não deve estar associada ao aumento da captação tecidual de glicose estimulada por insulina. O padrão metabólico apresentado por D. rotundus poderia estar associado a uma estratégia adaptativa, que garantiria maiores níveis de glicose circulante pelo maior tempo possível, já que esta espécie lida com a possibilidade constante de insucesso no forrageamento. Embora apresente fragilidade ao jejum, D. rotundus possui grande densidade populacional, o que poderia ter sido facilitado, em grande parte, pela existência do comportamento de compartilhamento recíproco do alimento, observado por Wilkinson (1984), em que um animal que não conseguiu se alimentar por uma noite pode receber sangue de outros morcegos da mesma unidade social, através da regurgitação. ________________________________________________________________________________________ ABSTRACT / In general, mammals fed on high protein diets (HP) are more resistant to starvation. However, the hematophagous bat Desmodus rotundus, althougth having a HP diet (blood), is markedly susceptive to the food deprivation, presenting severe hypoglycemia after 24h of starvation. Besides, this species has small stores of hepatic glycogen and adipose tissue lipid, low concentration of plasma and pancreatic insulin and a few numbers of á and â cells in its Islets of Langerhans. This work aimed to study in D. rotundus: the response to Intraperitonial Glucose Tolerance Test (ipGTT); Intraperitonial Insulin Tolerance Test (ipITT); static insulin secretion stimulated by many secretagogues; insulin tecidual signaling, and insulin glucagon and cortisol plasma levels in fed and fasted animals. The results from ipGTT showed that these bats cannot handle with an overload of glucose, suggesting intolerance to this substrate. The results from ipITT showed that there are not changes in glycemia after insulin injection suggesting that, besides the low insulin concentration, D. rotundus seems to present resistance to this hormone. Plasma glucagon and insulin concentrations of fed and starved bats were lower than most mammals, including humans. On the other side, the high levels of plasma cortisol suggest that this hormone seem not to contribute to glycemic homeostasis maintenance, as normally happens in mammals. The experiments with static insulin secretion showed that the â cells from D. rotundus presented a significant insulin secretion increase in response to glucose and leucina, but did not present to K+. Finally, we did not find changes in liver Akt phosphorilation levels in response to insulin stimulation. However, there was a significant increase in ERK expression in response to this hormone. Albeit we have found increase in ERK expression in response to insulin, this pathway should not be involved in glucose uptake stimulated by insulin. The metabolic pattern presented by D. rotundus could be related to an adaptative strategy, which guarantee high circulating glucose levels considering that these species deals with great possibility of not attaining to food. Although its fragility to starvation, D. rotundus shows a huge population density. This is possibly related to the reciprocal sharing food behavior (Wilkinson, 1984), where an animal that was not able to feed itself for one night receives blood from another bat by regurgitation.

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published_or_final_version / Zoology / Doctoral / Doctor of Philosophy

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A nonrecirculatory liver slice perifusion system has been developed and utilized for investigating glucagon-stimulated glycogenolysis in normal and diabetic states. It has been shown here that slices maintained in this system experience a controlled environment with respect to temperature and pH and remain viable throughout a three-hour experimental period based upon their maintenance of intracellular potassium levels. Although glycogen content falls by 40%, slices exhibit significant glycogenolysis in a dose-response manner upon challenge with glucagon, with maximal concentrations eliciting a 2.2-fold stimulation. This system, which permits nonrecirculatory challenge of liver tissue and subsequent analysis of both intracellular events and overall physiological responses, is extremely useful for examining hormonal mechanisms operating for glucagon, particularly at low concentrations. Using this methodology, liver slices challenged with glucagon exhibit a biphasic dose-response for glycogenolysis. While the second phase parallels cAMP (cyclic adenosine 3':5'-monophosphate) accumulation and cAMP-PK (cAMP-dependent protein kinase) activation, the first is mediated independent of cAMP. Trinitrophenylhistidine-1, homoarginine-12-glucagon (THG), which can antagonize glucagon-stimulated adenylate cyclase, exhibits 50% partial agonist activity for cAMP production and cAMP-PK but full agonism for glycogenolysis. Separation between these events is only two-fold indicating a cAMP-mediated process. [Des-amino-fYRKKE]-glucagon, ([Des-amino-His¹,D-Phe⁴,Tyr⁵,Arg¹²,Lys¹⁷·¹⁸,Glu²¹]-glucagon), another adenylate cyclase antagonist, does not stimulate cAMP or cAMP-PK up to 25 μM yet still elicits glycogenolysis. These results demonstrate that glucagon does indeed stimulate both cAMP-independent as well as cAMP-dependent glycogenolysis in normal liver. In diabetic systems, glucagon elicits attenuated adenylate cyclase activity in liver plasma membranes with reduction in basal activity and extent of stimulation. Maximal stimulation of cAMP production is also reduced by half in liver slices, but in both systems (normal vs. diabetic) EC₅₀ values for cAMP production are identical. Neither THG nor [des-amino-fYRKKE]-glucagon stimulate cAMP production or cAMP-PK in diabetic liver slices. While THG lowers blood glucose levels in vivo, [des-amino-fYRKKE]-glucagon acts as an agonist. These results suggest that the mechanisms which operate for glucagon-stimulated glycogenolysis in normal liver are attenuated in the diabetic state. Furthermore, antagonism of cAMP production alone is insufficient to antagonize glucagon's overall physiological action.