Thesis (MSc)--University of Stellenbosch, 2006. / ENGLISH ABSTRACT: Introduction: It is well documented that insulin offers cardioprotection against the consequences of ischaemia/reperfusion injury. Insulin-induced improvements in cardiac functions are widely investigated in models of ischaemia and reperfusion. It has been shown that many signalling pathways may be involved in the cardioprotection properties of insulin under those conditions. These pathways include PI3-K, PKB/Akt, p70S6k, ERK and many others. However, little data exists on the effects of insulin on the heart under normoxic condition. Some evidence has been presented that insulin has a positive inotropic effect on the normoxic perfused rat heart, but no precise cellular mechanism has been investigated or described in this regard. We believe that an investigation into the effects of insulin on cardiac function and pathways involved under normoxic conditions may help us to better understand the mechanisms of insulin-induced cardioprotection. Aims: To determine a suitable dose of insulin at which a positive inotropic response could be detectable under normoxic conditions, to investigate the possible mechanisms involved in insulin-induced increases in contractility with specific reference to the vasculature and the coronary flow and to investigate a possible involvement of PI3-K and its downstream effectors on the insulin effects on cardiac functions under normoxic conditions. Materials and methods: Isolated rat hearts were perfused retrogradely using the Langendorff technique. After 10 minutes of stabilization hearts were perfused for 30 minutes either with standard perfusion solution i.e. Krebs-Henseleit buffer + glucose gassed with 95%O2, 5%CO2 (control hearts), or with standard perfusion solution plus insulin alone or insulin together with the nitric oxide synthase inhibitor L-NAME or the PI3-K inhibitor wortmannin. Left ventricular developed pressure (LVDevP), heart rate (HR) and coronary flow (CF) as well as phosphorylated PI3-K and PKB/Akt in heart were measured. Results: Administration of insulin alone at physiological concentrations showed improved cardiac function compared to hearts in the control group. Hearts that received insulin+L-NAME showed a significant decrease in function compared to the control hearts and the hearts that received insulin alone (p<0.05). Phosphorylated PKB/Akt (Thr308) was increased in hearts that received insulin alone and insulin+L-NAME compared to the control hearts. Phosphorylated PI3-K tended to be higher in hearts where insulin was administered alone compared to the hearts that received insulin+L-NAME or insulin+wortmannin. Conclusion: This study confirmed that physiological concentrations of insulin exert positive inotropic effects on cardiac function in normoxic perfused rat hearts as seen with the improved LVDevP. Inhibition of PI3-K by wortmannin induced a decrease in phosphorylated PKB/Akt in hearts that received insulin+wortmannin and administration of L-NAME impaired the beneficial effects of insulin on cardiac functions. Therefore these results may indicate that nitric oxide may have a role in the positive effect of insulin on cardiac function in the healthy heart perfused under normoxic conditions. L-NAME as well as wortmannin reversed the positive inotropic effects of insulin. Both inhibitors also unmasked effects of insulin via nitric oxide and PI3-K on heart rate and coronary flow. / AFRIKAANSE OPSOMMING: Inleiding: Dit is welbekend dat toediening van insulien die hart beskerm teen ischemie/reperfusie-beserings, wat lei tot verbeterde hartfunksie. Hierdie effek word wyd ondersoek in modelle van ischemie en reperfusie. Dit is bewys dat ‘n verskeidenheid seintransduksie paaie, insluitend PI3-K, PKB/Akt, p70S6k en ERK, betrokke is by hierdie beskermende effek van insulien op die hart. Baie min data is egter beskikbaar rakende die effek van insulien tydens normoksiese toestande. Alhoewel dit bekend is dat insulien ’n inotropiese effek op die normale geperfuseerde hart het, is die presiese sellulêre meganismes wat dit bewerkstellig nog nie nagevors nie. Om dus ‘n beter begrip van hierdie meganismes te verkry is dit dus noodsaaklik om die effekte van insulien onder normoksiese perfusie toestande na te vors. Doelstellings: Om ‘n geskikte dosis, waarby insulien sy positiewe inotropiese effek onder normale toestande het, vas te stel, om die moontlike meganismes betrokke by insulien-geïnduseerde verbetering in hartsametrekbaarheid te bestudeer, met spesifieke verwysing na die bloedvoorsiening en koronêre vloei, en om die moontlike betrokkenheid van die PI3-K pad en sy teiken effektore onder normale suurstof-toestande te ondersoek. Materiaal en metodes: Geïsoleerde rotharte is geperfuseer deur gebruik te maak van die Langendorff tegniek. Na ‘n stabilisasie periode van 10 minute is rotharte blootgestel aan 30 minute perfusie met een van vier oplossings: ‘n standaard perfusie oplossing (Krebs-Henseleit buffer met glukose onder spesifieke gaskondisies van 95% O2, 5% CO2 – kontrole harte); standaard perfusie oplossing en insulien; standaard perfusie oplossing met insulien en die stikstofoksied sintase inhibitor L-NAME, of standaard perfusie oplossing, met insulien en die PI3-K inhibitor wortmannin. Met verloop van die perfusie protokol, is ontwikkelde linker ventrikulêre druk (LVDevP), harttempo (HR) en koronêre vloei (CF), sowel as PI3-K en PKB/Akt fosforilasie, gemeet. Resultate: Toediening van insulien teen fisiologiese konsentrasies het ‘n verbeterde hartfunksie tot gevolg, in vergelyking met harte in die kontrole groep. In teenstelling hiermee het harte wat insulien+L-NAME ontvang het ‘n betekenisvolle verlaagde funksie getoon in vergelyking met die kontrole harte en harte wat slegs insulien ontvang het (p<0.05). Harte wat slegs insulien, of insulien+L-NAME ontvang het, het ‘n verhoging in gefosforileerde PKB/Akt (Thr308) getoon in vergelyking met kontrole harte. Gefosforileerde PI3-K het ook geneig om hoër te wees in harte wat insulien+L-NAME of insulien+wortmannin ontvang het, as in harte wat slegs insulien ontvang het. Gevolgtrekking: Hierdie studie bewys dat fisiologiese konsentrasies van insulien, onder normale suurstof-toestande, ‘n positiewe inotropiese effek op hartfunksie uitoefen, soos gesien in die verbeterde LVDevP. Wortmannin-geïnduseerde inhibering van die PI3-K pad het ‘n verlaagde PKB/Akt fosforilasie tot gevolg gehad in harte wat insulien+wortmannin ontvang het, terwyl die toediening van L-NAME die voordelige effekte van insulien op hartfunksie onderdruk het. Hierdie resultate dui dus aan dat stikstofoksied ‘n rolspeler is in die positiewe inotropsiese effek van insulien op hartfunksie tydens normoksiese toestande, aangesien beide inhibitore hierdie effek onderdruk het. Beide inhibitore het ook die betrokkenheid van stikstofoksied en die PI3-K pad by die effek van insulien op harttempo en koronêre vloei onthul.

The ability of insulin to stimulate glucose uptake into muscle and adipose tissue is central to the maintenance of whole-body glucose homeostasis. Deregulation of insulin action manifests itself as insulin resistance, a key component of type 2 diabetes. Insulin resistance is also observed in HIV patients receiving protease inhibitors. An agent that can reversibly induce an insulin-resistant state would be a very useful tool in developing model systems that mimic the pathogenesis of type 2 diabetes. Insulin resistance can arise from defects in insulin signal transduction, changes in the expression of proteins or genes that are targets of insulin action, cross talk from other hormonal systems or metabolic abnormalities. Deterioration of the insulin-receptor-signalling pathway at different levels leading to decreased levels of signalling pathway intermediates and/or decreased activation through phosphorylation accounts for the evolution from an insulin-resistant state to type 2 diabetes. In addition, defects in GLUT4 glucose transporter translocation are observed, further fuelling impairments in skeletal muscle glucose uptake. Levels of insulin-induced GLUT4 translocation in the skeletal muscle of type 2 diabetic patients are typically reduced by 90%. Many cellular pathways & their intermediates are in some way or another linked to insulin signalling. This study focused on three of these namely the PI3-kinase/Akt pathway, the Mitogen Activated Protein Kinase (MAPK) cascade and the AMP Kinase pathway, with successful monitoring of the PI3-K pathway. Investigations involved observing and evaluating the effects of various compounds as well as an indigenous medicinal plant, Sutherlandia frutescens on the activities of key insulin signalling pathway intermediates within the three fore mentioned pathways including Akt, AMPK and MEK1/2 as well as membrane surface GLUT4 levels. Scientific research has in the past leant heavily on Western blotting as the method of choice for gaining vital information relating to signal transduction pathways, however for research into cellular mechanisms the negatives of this method outweigh the positives. The drawbacks include a need for large amount of cells, multiple washing steps which may be disadvantageous to any weak and transient interactions as well as lysing of cells which may interfere with the maintenance of the subcellular localisation of a specific signalling event. Based on these, the need for a better method in terms of speed & reliability to monitor phosphorylation states of signal transduction pathway intermediates & GLUT4 translocation was evident and was one VII of the main aims & successes of this study. The method created used the mouse muscle cell line C2C12 in conjunction with the quick, sensitive method of flow cytometry which allowed us to monitor these processes in these cells through immune-labelling. Adherent cell cultures such as the C2C12 cell line pose the problem of possible damage to plasma membrane receptors (including insulin receptors) during harvesting to obtain a cell suspension for flow cytometry. We however used C2C12 mouse myocytes to optimize a method yielding insulin responsive cells in suspension that were successfully used for flow cytometry after immunelabelling of insulin signalling intermediates. Insulin (0.1μM) significantly raised the levels of both P-Akt and GLUT4 above basal levels. This effect was shown to be dose dependent. At a concentration of 50μg/ml, Sutherlandia frutescens was able to act as an insulin-mimetic in terms of its ability to increase P-Akt levels, GLUT4 translocation and glucose utilisation in an acute manner. These increases could be reduced with the addition of wortmannin, a PI3-K inhibitor. Therefore, these results suggest the mechanism of the plant extract’s insulin-like activity may be in part due to the activation of the insulin signalling pathway leading to GLUT4 translocation, which involves the phosphorylation of insulin receptor- and subsequent PI3-K activity, leading to P-Akt activity. These results provide further evidence of this plant extract’s anti-diabetic potential. The effect of Sutherlandia frutescens on insulin secretion, calcium signalling and proliferation in INS-1 rat pancreatic cells was also investigated and it was found to increase the activities of all of these processes. However no change in the levels of GLUT2 glucose transporter was seen. Ritonavir is prescribed by the South African Department of Health in co-formulation with other protease inhibitors within its second regime in the treatment of HIV and AIDS. Using C2C12 cells, ritonavir decreased glucose uptake acutely and had no effect on GLUT4 translocation however surprisingly increased P-Akt levels. In conclusion, it was found that Sutherlandia frutescens has antidiabetic benefits, diverse in nature depending on tissue type as well as length of time administered. The establishment of novel flow cytometry techniques to assess antidiabetic properties using in vitro cell culture was achieved. These methods will be useful in the future for the assessment of insulin sensitivity and in the identification of novel compounds that stimulate the insulin signalling pathways.

Diss. (sammanfattning) Linköping : Univ., 2001. / Härtill 5 uppsatser.