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Call number: LD2668 .T4 1966 F854 / Master of Science

The purpose of this investigation was to determine the relationship between calcium and glucose uptake following muscle contraction with the use of the calcium channel blocker dantrolene. In previous studies an exercise model has been used to investigate the role of calcium during post-exercise glucose uptake. This study utilized electrical stimulation. It has been shown that exercise-induced glucose uptake is calciummediated, but to date no one has shown that glucose transport induced by electrical stimulation is calcium-mediated. Twenty four male Sprague Dawley rats weighing 140 g were sacrificed and their epitrochlearis muscles were removed. Four treatment groups were established: control, muscle incubated in glucose (4mM); insulin, muscles incubated in glucose (4mM) and insulin (1000uU/ml); electrical stimulation, at 50 Hz for two five minute intervals separated by one minute rest periods; insulin (1000uU/ml) and electrical stimulation at 50 Hz for two five minute intervals separated by one minute intervals. Each group consisted of contain 8-10 muscle preparations. Glucose uptake was measured through the use of a double label of radioactive mannitol and 3-O-methylglucose and analyzed using liquid scintillation. This project followed a randomized group design. Treatments were measured with a one way ANOVA. / School of Physical Education

Thesis (MSc)--Stellenbosch University, 2001. / ENGLISH ABSTRACT: Introduction: It is well documented that insulin offers cardioprotection under ischaemic stress. In the past it was believed that the protective effects of insulin, such as the (a) recruitment of glucose transporters to enhance glucose entry into the cell, (b) stimulation of glycolysis, (c) enhancement of glycogen synthesis, (d) improved protein synthesis, and (e) positive inotropic and chronotropic properties, were metabolic of origin, but lately the emphasis has shifted towards the diverse signal transduction pathways elicited by insulin. Although these beneficial effects of insulin on ischaemia/reperfusion induced injury have been studied for many years, the exact protective mechanism is still not resolved. Aim: To investigate the influence of insulin on the signaling pathways as a possible protective mechanism against ischaemia/reperfusion and therefore to investigate the possible roles and cross signaling of cyclic adenosine monophosphate (cAMP), protein kinase B (PKB) and p38 mitogen activated protein kinase (p38 MAPK) in the cardioprotection offered by insulin to the reperfused, ischaemic myocardium. Materials and methods: Isolated rat hearts were perfused retrogradely in accordance with the Langendorff technique (95%02, 5% C02). After 30 min of stabilization, hearts were subjected to 30 min global low flow ischaemia (0,2 ml/min), followed by 30 min of reperfusion. Hearts perfused with standard Krebs Henseleit solution containing 5 mM glucose were compared to hearts perfused with a perfusion solution containing 5 mM glucose and 0,3 IlIU/ml insulin. Wortmannin was added during either ischaemia or reperfusion. Left ventricular developed pressure (LVDP), rate pressure product (RPP), tissue cAMP and PKB and p38 MAPK activation were measured. Results: Insulin treated hearts showed improved functional recovery (P<0.05) during reperfusion after ischaemia vs. non-insulin treated hearts (85.5±4.6% vs. 44.8±4.9%). However, the addition of wortmannin (a Pl3-kinase inhibitor) to the perfusion solution during either ischaemia or reperfusion abolished the improved recovery. At the end of ischaemia, cAMP levels of the insulin treated hearts were elevated significantly, while the cAMP content in the non-insulin treated hearts returned to control levels. Addition of wortmannin during ischaemia abolished this rise in cAMP. Wortmannin added during reperfusion only did not alter the levels of cAMP at the end of reperfusion. Activation of p38 MAPK was transient during ischaemia for both insulin and non-insulin treated hearts. Addition of wortmannin during ischaemia did not alter p38 MAPK levels at the end of ischaemia. P38 MAPK was activated significantly (P<0.001) in the non-insulin treated hearts vs. insulin treated hearts during reperfusion. Wortmannin, added at the onset of reperfusion, could partially abolish the effects of insulin to suppress p38 MAPK activation after 30 min of reperfusion. Activation of PKB in insulin treated hearts was significantly higher than non-insulin treated hearts during stabilization and early ischaemia. This activity was depressed by 30 min of ischaemia in both presence and absence of insulin. Wortmannin, when added before induction of ischaemia did not further lower this. The presence of insulin resulted in occurrence of strong PKB activation during reperfusion, peaking at 15 minutes and diminishing at 30 minutes. Wortmannin, added at the onset of reperfusion, abolished PKB activity measured at the end of reperfusion. Conclusion: Insulin exerted a positive inotropic effect and delayed the onset to ischaemic contracture. Inhibition of Pl3-kinase by wortmannin abolished the protective effects of insulin, arguing for an insulin stimulated PKB involvement in cardiac protection. Insulin also increased cAMP production and attenuated activation of p38 MAPK, both associated with improved recovery. This evidence suggested possible cross signaling between different signaling pathways. / AFRIKAANSE OPSOMMING: Agtergrond: Insulin beskerm harte wat aan isgemiese stres blootgestel word. Alhoewel hierdie voordelige effekte van insulien reeds vir verskeie jare bestudeer is, is die presiese meganisme waarmee insulien die hart beskerm steeds nie duidelik nie. Navorsers het die beskermende effekte van insulien aan metaboliese gevolge soos: (a) verhoogde glukose transport d.m.v. inspanning van meer glukose transporters (b), stimulering van glikolise, (c) vebeterde glikogeensintese, (d) verhoogde proteiensintese, en (e) die positiewe inotropiese en chronotropiese eienskappe van insulien toegeskryf. Onlangs het die fokus verskuif na ander diverse seintransduksiepaaie. Doel: Die doel van hierdie studie was dus om die moontlike betrokkenheid van hierdie sientransduksiepaaie asook die interaksie tussen sikliese adenomonofosfaat (cAMP), proteïn kinase B (PKB) en p38 MAPK in die beskerming wat insulien aan die isgemiese, gereperfuseerde miokardium bied, te bestudeer. Materiale en Metodes: Geïsoleerde rotharte is geperfuseer in ooreenstemming met die Langendorff metode. Na 30 min van stabilisasie is harte blootgestel aan 30 min. globale lae vloei isgemie (0,2 ml/min), en daarna is harte vir 30 min. geherperfuseer. Harte wat geperfuseer is met 'n perfusaat wat 5mM glukose bevat is vergelyk met harte wat geperfuseer is met 'n perfusaat wat 5mM glukose en 0,3 ~IU/ml insulien bevat. Sommige harte is geperfuseer met 'n perfusie oplossing waar wortmannin bygevoeg is tydens óf isgemie óf tydens herperfusie. Linker ventrikulêre ontwikkelde druk (LVDP), tempo-druk produk (RPP), weefsel cAMP-vlakke asook PKB en p38 MAPK aktiwiteit is gemeet. Resultate: Insulien-behandelde harte het funksioneel beduidend beter herstel tydens herperfusie na isgemie as harte wat nie met insulien behandel is nie (85.5±4.6% vs. 44.8±4.9%). Byvoeging van wortmannin by die perfusie oplossing tydens óf isgemie óf reperfusie, het die toename in herstel wat gesien is in die insulien-behandelde harte, opgehef. Die cAMP vlakke in die insulienbehandelde harte het aan die einde van isgemie beduidend gestyg (P<0.001), terwyl vlakke in harte wat nie met insulien behandel is nie, na kontrole vlakke teruggekeer het. Die teenwoordigheid van wortmannin in die perfusie oplossing tydens isgemie, het die styging in cAMP voorkom , terwyl die byvoeging van wortmannin tydens herperfusie. nie die cAMP vlakke beïnvloed het nie. Die aktivering van p38 MAPK tydens isgemie was van verbygaande aard in beide die insulien-behandelde harte en harte wat nie met insulien behandel is nie. Die byvoeging van wortmannin tydens isgemie het nie die p38 MAPK aktivering beïnvloed nie. P38 MAPK is beduidend geaktiveer tydens herperfusie in harte wat nie met insulien behandel is nie vergeleke met die insulien-behandelde harte. Die byvoeging van wortmannin tydens reperfusie kon die effek van insulien om p38 MAPK aktivering te onderdruk, gedeeltelik ophef. PKB aktivering tydens die stabilisasie fase en vroeë isgemie was beduidend hoër in die insulien-behandelde harte vs. die harte wat nie met isulien behandel is nie. Die aktiwiteit is onderdruk deur 30 min isgemie ongeag die teenwoordigheid van insulien. Die byvoeging van wortmannin tydens isgemie het PKB aktivering nie verder verlaag nie. Die teenwoordigheid van insulien het 'n sterk aktivering van PKB tydens herperfusie veroorsaak met 'n piek na 15 min en 'n verlaging na 30 min. Wortmannin bygevoeg aan die begin van herperfusie, het PKB aktiwiteit opgehef aan die einde van reperfusie. Opsomming: Insulien het 'n positiewe inotropiese invloed gehad, en het die begin van isgemiese kontraksie vertraag. Die inhibisie van Pl3-kinase deur wortmannin het die beskermende effekte van insulin opgehef, wat 'n insulin gestimuleerde PKB betrokkenheid aandui. Insulien het ook verhoogte cAMP produksie en verlaagde p38 MAPK aktivering tot gevolg gehad, en beide is geassosieer met verbeterde herstel. Hierdie resultate dui dus op moontlike interaksie tussen die verskillende seintransduksiepaaie.

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published_or_final_version / Molecular Biology / Master / Master of Philosophy

Orientador: Dora Maria Grassi-Kassisse / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-14T17:17:00Z (GMT). No. of bitstreams: 1 Martinelli_TiagoCastrodePadua_M.pdf: 1034974 bytes, checksum: 979cea9aa8b38892d83b77fff6b500ad (MD5) Previous issue date: 2009 / Resumo: Está bem descrito na literatura que o exercício físico aeróbio estimula a maior captação de glicose predominantemente no músculo esquelético e que o tecido adiposo branco também participa do aumento da captação de glicose estimulada pela insulina em indivíduos exercitados. O objetivo deste trabalho foi avaliar a sensibilidade à insulina em adipócitos de ratos submetidos ao modelo de natação implantado no Labeest. Ratos Wistar adultos foram submetidos à sessões diárias de natação (50 min, 5 vezes por semana, durante 28 dias) e foi observado que ao final do protocolo experimental os ratos apresentaram redução do ganho de peso corpóreo e do consumo médio de ração. O ensaio de clamp euglicêmico-hiperinsulinêmico demonstrou, através taxa de infusão de glicose (GIR), que a natação induziu maior sensibilidade dos tecidos à insulina (C: 509,2 ± 30,5 mg/Kg/min; N: 679,1 ± 26,6 mg/Kg/min). As análises de quantificação de expressão total das proteínas GLUT 4 e AMPK em tecido adiposo isolado mostraram um aumento significativo apenas no GLUT 4. A quantidade de lactato basal produzida pelos adipócitos isolados de ratos exercitados foi significativamente maior que aquela observada nos ratos controle (C: 0,1977 ± 0,0640 µmol lactato.106 cels/60 min; N: 0,2818 ± 0,0454 µmol lactato.106 cels/60 min). Além disso, os agonistas não aumentaram a produção de lactato nos adipócitos isolados de ratos exercitados. O prazosin aumentou significativamente a produção de lactato pela ação da noradrenalina, em adipócitos de ratos exercitados (C: 0,9596 ± 0,2490 µmol lactato.106 cels/60 min; N: 1,2180 ± 0,3055 µmol lactato.106 cels/60 min). Por outro lado o protocolo de natação não alterou a produção e liberação de glicerol basal (C: 0,0716 ± 0,0207 µmol glicerol 106 cels/60 min) ou estimulada, enquanto o prazosin inibiu a produção de glicerol pela noradrenalina (C: 0,5132 ± 0,1023 ; N: 0,4681 ± 0,0331 µmol glicerol 106 cels/60 min) em adipócitos isolados desses animais. O exercício de natação, conforme modelo proposto, promove aumento na captação de glicose devido a uma maior expressão de transportadores GLUT 4, sendo que esta glicose, captada no adipócito, constituiu-se em fonte para a produção de lactato, uma vez que a via lipolítica não foi alterada. / Abstract: It is well described in the literature that aerobic physical exercise stimulate glucose uptake and this is mainly associated with skeletal muscle. However, it has been related that white adipose tissue also contributes to insulin increase glucose uptake after the physical training. The aim of this work was to evaluate insulin sensitivity in rats white adipocytes submitted to the model of swimming proposed to Labeest. Adult Wistar rats had been submitted to the daily sessions of swimming (50 min, 5 times per week, during 28 days). At the end of protocol rats that had swum presented reduction of the profit of corporeal weight and the average consumption of chow. The euglycemic-hyperinsulinemic clamp assays demonstrated that swimming induced increase in tissue insulin sensitivity (C: 509.2 ± 30.5 mg/Kg/min; N: 679.1 ± 26.6 mg/Kg/min). The analyses of quantification of total expression of proteins GLUT 4 and AMPK in adipose tissue had shown a significant increase only in GLUT 4. The amount of basal lactate produced by the isolated adipocytes of exercised rats was significantly higher that observed in control rats (C: 0.1977 ± 0.0640 µmol lactato.106 cels/60 min; N: 0.2818 ± 0.0454 µmol lactato.106 cels/60 min). Moreover, the agonists had not increased the lactate production in the isolated adipocytes of exercised rats. Prazosin significantly increased the lactate production stimulated by noradrenaline in adipocytes of exercised rats (C: 0.9596 ± 0.2490 µmol lactato.106 cels/60 min; N: 1.2180 ± 0.3055 µmol lactato.106 cels/60 min). On the other hand the swimming protocol did not modify the production of basal (C: 0.0716 ± 0.0207 µmol glycerol 106 cels/60 min) or stimulated release of glycerol, while prazosin inhibited the production of glycerol induced by the noradrenaline (C: 0.5132 ± 0.1023 µmol glycerol 106 cels/60 min; N: 0.4681 ± 0.0331 µmol glycerol 106 cels/60 min) in isolated adipocytes of these animals. Concluding, the exercise of swimming in the considered model increased the glucose uptake due to a higher expression of transporters GLUT 4. This glucose, once in the adipocytes, seems to be the main source for the lactate production because the lipolytic way was not modified. / Mestrado / Fisiologia / Mestre em Biologia Funcional e Molecular

Indiana University-Purdue University Indianapolis (IUPUI) / In response to glucose, pancreatic islet beta cells secrete insulin in a biphasic manner, and both phases are diminished in type 2 diabetes. In beta cells, cortical F-actin beneath the plasma membrane (PM) prevents insulin granule access to the PM and glucose stimulates remodeling of this cortical F-actin to allow trafficking of insulin granules to the PM. Glucose stimulation activates the small GTPase Cdc42, which then activates p21-activated kinase 1 (PAK1); both Cdc42 and PAK1 are required for insulin secretion. In conjunction with Cdc42-PAK1 signaling, the SNARE protein Syntaxin 4 dissociates from F-actin to allow SNARE complex formation and insulin exocytosis. My central hypothesis is that, in the pancreatic beta cell, glucose signals through a Cdc42-PAK1-mediated pathway to remodel the F-actin cytoskeleton to mobilize insulin granules to SNARE docking sites at the PM to evoke glucose stimulated second phase insulin secretion. To investigate this, PAK1 was inhibited in MIN6 beta cells with IPA3 followed by live-cell imaging of F-actin remodeling using the F-actin probe, Lifeact-GFP. PAK1 inhibition prevented normal glucose-induced F-actin remodeling. PAK1 inhibition also prevented insulin granule accumulation at the PM in response to glucose. The ERK pathway was implicated, as glucose-stimulated ERK activation was decreased under PAK1-depleted conditions. Further study showed that inhibition of ERK impaired insulin secretion and cortical F-actin remodeling. One of the final steps of insulin secretion is the fusion of insulin granules with the PM which is facilitated by the SNARE proteins Syntaxin 4 on the PM and VAMP2 on the insulin granule. PAK1 activation was also found to be critical for Syntaxin 4-F-actin complex dynamics in beta cells, linking the Cdc42-PAK1 signaling pathway to SNARE-mediated exocytosis. Syntaxin 4 interacts with the F-actin severing protein Gelsolin, and in response to glucose Gelsolin dissociates from Syntaxin 4 in a calcium-dependent manner to allow Syntaxin 4 activation. Disrupting the interaction between Syntaxin 4 and Gelsolin aberrantly activates endogenous Syntaxin 4, elevating basal insulin secretion. Taken together, these results illustrate that signaling to F-actin remodeling is important for insulin secretion and that F-actin and its binding proteins can impact the final steps of insulin secretion.