Modelling the insulin signaling pathway and glucose transport induction in differentiated 3T3-L1 mouse adipocytes

Myburgh, Lauren (2021-04)

Thesis (MSc)--Stellenbosch University, 2021.

Thesis

ENGLISH ABSTRACT: Insulin is the principal controller of energy homeostasis in the human body. It lowers postprandial blood glucose and enforces a change in cellu- lar metabolism to maintain glucose homeostasis by stimulating glucose uptake into muscle and adipose tissue. Dysfunction in this response can lead to insulin resistance which is a manifestation of type 2 diabetes for which the molecular mechanisms are not well understood. Although adipose tissue contributes less to peripheral glucose utilization compared to muscle tissue, knockout studies have identified adipose tissue as a key component in the dysfunction of insulin signalling. With the prevalence of this disease increasing drastically world- wide and majority of these patients being overweight, adipose tissue can be regarded as unhealthy despite its many health benefits, which include regu- lating energy balance and communicating with other organs as it functions as an endocrine organ. Therefore, it is crucial to firstly understand the ref- erence state under which the insulin signalling network and glucose uptake occurs as there is difficulty in identifying key components that result in type 2 diabetes. The network originates with the activation of the insulin receptor (IR) by insulin before eliciting a wide range of cellular responses that together coordinate glucose uptake into adipose tissue. Therefore, the primary purpose of this study was to describe the interactions involved between insulin and the intermediates, IR and Protein Kinase B (Akt) in the insulin signalling network and the e ect thereof on glucose uptake and metabolism in adipose tissue under a reference state using a systems biology approach. Firstly, the adipose tissue used in this study was 3T3-L1 adipocytes. Secondly, the intermediates, IR and Akt, were analyzed using Western blot analysis and glucose transport activity was measured using radio-labelled glucose uptake using these di erentiated 3T3-L1 adipocytes. Finally, the ux through glycolysis was measured to determine whether the transport activity changes in the same degree to the ux. The response of the insulin signalling network intermediates and glucose transport activity in a dose-dependent and time responsive manner were determined. This resulted in a hyperbolic relationship between insulin and the IR over time, demonstrated by both the experimental results and the mathematical model used. Experimentally an overshoot response, which is de ned as transient peaks of phosphorylation followed by a lower steady state level, was observed between insulin and Akt. The lower steady state level was caused by a down-regulation of the signal while still in the presence of insulin. However, the model could not predict this response as the molecular mechanisms that result in the overshoot behaviour are not known and therefore were not included in the model restricting the system to a hyperbolic response. The model could to some extent, however, predict the time responsive phosphorylation and dephosphorylation dynamics of the interactions involved. Thus, the study was able to capture the dynamic behaviour of the system in question thereby allowing a better understanding of the interactions between insulin and its intermediates and its a ect on glucose transporter activity and metabolism within a reference state in 3T3-L1 adipocytes.

AFRIKAANSE OPSOMMING: Insulien is een van die hoofkomponente in die beheer van energie-homeostase in die menslike liggaam. Na ete word bloedglukosevlakke deur insulien verlaag en veroorsaak dit 'n verandering in sellulêre metabolism spesi ek om glukose-homeostase te kan handhaaf, deur die opname van glukose in spier- en vetweefsel te stimuleer. Insulienweerstandigheid is die gevolg van 'n disfunksie van hierdie reaksie en manifesteer as tipe 2-diabetes. 'n Begrip van die molekulêre meganismes van hierdie reaksie is steeds onvolledig. Alhoewel vetweefsel minder bydra tot die gebruik van glukose in die periferie in vergelyking met spierweefsel, het uitklopstudies vetweefsel geïdenti seer as 'n belangrike komponent in die disfunksie van insulien seinmeganismes. Met die drastiese toename in wêreldwye voorkoms van hierdie siekte, terwyl die meerderheid van hierdie pasiënte oorgewig is, kan vetweefsel as ongesond beskou word ondanks die verskeie gesondheidsvoordele wat dit moontlik inhou. Hierdie voordele sluit in regulering van energiebalans en die gevolglike kommunikasie met ander or-gane. Dit is van kardinale belang om eers 'n verwysingstoestand waaronder die insuliennetwerk en die opname van glukose plaasvind, te verstaan juis omdat dit so uitdagend is om die hoofkomponente te identi seer wat tipe 2-diabetes tot gevolg het. Die netwerk begin met die aktivering van die insulienreseptor (IR) deur insulien wat 'n verskeidenheid sellulêre reaksies tot gevolg het wat die opname van glukose in vetweefsel koördineer. Die primêre doel van hierdie studie was dus om 'n sisteembiologie-benadering te gebruik om die interaksies tussen insulien en die tussenprodukte, IR en proteïenkinase B (Akt) van die insulien-seinnetwerk en die e ek daarvan op glukose-opname en metabolisme in vetweefsel onder 'n verwysingstoestand, te beskryf. Die vetweefsel wat in hierdie studie gebruik is, is 3T3-L1 vetselle. Die tussenprodukte, IR en Akt, is geanaliseer met behulp van Western blot-ontleding en glukosetransport aktiwiteit is met behulp van radio-gemerkte glukose-opname, deur die gebruik van gedi erensieerde 3T3-L1-vetselle, gemeet. Laastens, is die uksie deur glikolise gemeet om te bepaal of transport aktiwiteit met dieselfde graad van verandering as die uksie plaasgevind het. Die respons van die tussenprodukte van die insulienseinnetwerk en glukosetransport aktiwiteit is in 'n dosisafhanklike en tyd-afhanklike manier bepaal. Dit het 'n hiperboliese verband tussen insulien en die IR oor tyd tot gevolg gehad, wat deur die eksperimentele resultate en die wiskundige model wat in die studie gebruik is, getoon word. Eksperimenteel is 'n oorskietreaksie, wat beskryf kan word as 'n skielike toename in fosforilasie gevolg deur 'n laer bestendige toestand, tussen insulien en Akt waargeneem. Die laer bestendige toestand is veroorsaak deur 'n afname in regulering van die sein terwyl dit steeds in die teenwoordigheid van insulien is. Die model kon hierdie reaksie egter nie voorspel nie, aangesien die molekulêre meganismes wat die oorskietgedrag tot gevolg het, nie bekend is nie en kon dus nie in die model ingevoer word nie, wat die sisteem beperk tot 'n hiperboliese verhouding. Die model kon egter tot 'n sekere mate die tyd-afhanklike responsiewe fosforilasie en defosforilasie dinamika van die betrokke interaksies voorspel. Hierdie studie was in staat om dinamiese en insetafhanklike gedrag van die betrokke sisteem vas te lê en sodoende 'n beter begrip van die interaksies tussen insulien en die tussenprodukte asook die invloed daarvan op glukosetransport aktiwiteit en metabolisme binne 'n verwysingstoestand in 3T3-L1 vetselle, te bewerkstellig.

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