Quantification of the insulin response in rat L6 skeletal muscle cells

Shaw, Klarissa (2020-04)

Thesis (MSc)--Stellenbosch University, 2020.

Thesis

ENGLISH ABSTRACT: Mathematical models can help us to understand complex molecular mechanisms underlying diseases. There is a lack of these types of models describing the cellular processes involved in conditions such as insulin resistance and Type 2 diabetes (T2D). Therefore, this study aimed to quantify the reference state of the insulin signalling pathway for the construction of a mathematical model describing the dynamics of the pathway intermediates. This model will serve as a comparison for a model describing the diabetic state. Western blot analysis was used to quantify the phosphorylation states of Akt Ser473 and Thr308 at varying concentrations of insulin and over time, with or without insulin. The glucose transporter, GLUT4, activity was also investigated at deferent concentrations of insulin. These data were used in the construction of a model for the reference state. Ordinary deferential equations (ODEs) were derived to describe the dynamics of Akt over time. Steady state constraints were used to _t the dose response for Akt and made it possible to estimate a dephosphorylation/phosphorylation ratio. The estimated ratio was used to determine if the model could describe the time course data. A similar analysis was done for GLUT4. The model was able to accurately describe the phosphorylation and dephosphorylation dynamics of the Akt Ser473 and Thr308 site. In order to determine the link between insulin signalling and glucose transport, transport activity was plotted against Akt phosphorylation and the model was fitted to this data. The _t for both the Ser473 and Thr308 site did not differ. We found that the model predictions correlated quite well with the experimental data despite the lack of data on intermediates between insulin and Akt.

AFRIKAANSE OPSOMMING: Wiskundige modelle kan ons help om komplekse molekulêre meganismes onderliggend aan siektes te verstaan. Daar is 'n gebrek aan hierdie tipe modelle wat die sellulêre prosesse beskryf wat betrokke is by toestande soos insulienweerstandigheid en Tipe 2 diabetes (T2D). Daarom het hierdie studie ten doel gehad om die verwysingstoestand van die insulien seinepad te kwanti_seer vir die konstruksie van 'n wiskundige model wat die dinamika van die tussengangerselprodukte beskryf. Hierdie model sal as vergelyking dien vir 'n model wat die diabetiese toestand beskryf. Western blot analise is gebruik om die fosforileringstoestande van Akt Ser473 en Thr308 te kwanti_seer by verskillende konsentrasies insulien en met verloop van tyd, met of sonder insulien. Die glukosetransporter, GLUT4, is ook by verskillende konsentrasies insulien ondersoek. Hierdie is gegewens gebruik vir die konstruksie van 'n model vir die verwysingstoestand. Gewone di_erensiaalvergelykings (ODE's) is afgelei om die dinamika van Akt oor tyd te beskryf. Gereelde beperkings is gebruik om by die dosisrespons vir Akt te pas en het dit moontlik gemaak om 'n defosforilering / fosforilasie-verhouding te skat. Die beraamde verhouding is gebruik om te bepaal of die model die tydsverloopdata kon beskryf. 'n Soortgelyke analise is vir GLUT4 gedoen. Die model kon die fosforilering en defosforilering dinamika van die Akt Ser473 en Thr308 akkuraat beskryf. Ten einde die verband tussen insulien seine en glukose vervoer te bepaal, is vervoeraktiwiteit teen Akt fosforilering geplot en die model was op hierdie date gepas. Die pas vir beide die Ser473 en Thr308 verhouding met GLUT4 het nie verskil nie. Ons het gevind dat die modelvoorspellings redelik goed met die eksperimentele data gekorreleer het, ondanks die gebrek aan data oor tussenprodukte tussen insulien en Akt.

Please refer to this item in SUNScholar by using the following persistent URL: http://hdl.handle.net/10019.1/108463
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