Mathematical modelling of glycolysis in skeletal muscle cells
Date
2020-04
Authors
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Publisher
Stellenbosch : Stellenbosch University
Abstract
ENGLISH ABSTRACT: Diabetes is a term used to describe a group of metabolic diseases characterised by high blood glucose levels. Regulation of blood glucose levels is facilitated by the insulin stimulation of target tissues, such as skeletal muscle tissue and fat cells. A decrease in the effective working of insulin on target tissues can lead to a variety of symptoms, including damage or dysfunction of organs, such as the eyes, kidneys, nerves, and the heart and ultimately leads to the development of type 2 diabetes. Blood glucose homeostasis is predominately regulated by the metabolic activity of skeletal muscle fibres (70 – 80%). Therefore, there is an interest to investigate the dysfunctions caused by insulin resistance in skeletal muscle cells. In this thesis we present the kinetic characterisation of the glycolytic enzymes in C2C12 skeletal muscle fibres. The kinetic rate equations, describing the dynamic behaviour of each enzyme, was compiled into a kinetic model. The development of a novel HPLC analytical technique for the separation and quantification of glycolytic intermediates and cofactors was also presented in the thesis. This HPLC technique enables the separation and quantification of the glycolytic cofactors; ATP, ADP, AMP, NAD+ and NADH via UV/Vis detection, along with the separation and quantification of 13 glycolytic species derived from glucose, including ACA and ETOH, as well as GLY and G3P via the use and detection of 14C-radioactive labelling. The novel HPLC technique was used to validate the kinetic model describing glycolysis in C2C12 skeletal muscle cells by comparing model simulations to experimental data. 3 incubations of C2C12 skeletal muscle extract was analysed under different concentrations of GLC, FBP, ATP and NAD+ via the HPLC technique and compared to the corresponding model simulations. A good comparison between experimental data and model simulations was obtained, validating the glycolytic model describing glycolysis in C2C12 skeletal muscle extract. Metabolic control analysis was performed on the validated model and showed that the majority of flux control was held by the demand for ATP, described by the ATPase reaction (99.4%). This is in comparison with what is found in literature.
AFRIKAANSE OPSOMMING: Diabetes is `n term wat gebruik word om `n groep metaboliese siektes, gekenmerk deur hoë bloed glukose vlakke te beskryf. Regulasie van bloed glukose vlakke word gehandhaaf deur die werking van insulien op teiken weefsels, soos spierweefsel en vetselle. `n Afname in the effektiewe werking van insulin op teikenweefsels lei tot `n wye verskeidenheid van simptome, insluitend skade of versaking van organe soos die oë, niere, senuwees en die hart en lei tot die ontwikkeling van tipe 2 diabetes. Die regulasie van bloed glukose homeostase word oorwegend deur die metaboliese aktiwiteit van skelet spierweefsel (70 – 80%) genhandhaaf. Daarom is daar groot belang in die ondersoek van die versaking van insulin werking, afkomstig van insulien weerstandbediendheid in skelet spierweefsel. In hierdie tesis lê ons die biochemiese karakterisering van die glikolitiese ensieme in C2C12 skelet spiervesels voor. Die kinetiese tempovergelykings, wat die dinamiese gedrag van elke ensiem beskryf, was in `n kinetiese model saamgestel. Die ontwikkeling van `n nuwe HPLC analise tegniek vir die skeiding en kwantifisering van glikolitiese intermediate en kofaktore in selekstrak word ook in hierdie tesis voorgelê. Dié HPLC tegniek handhaaf die skeiding en kwantifisering van die glikolitiese kofaktore; ATP, ADP, AMP, NAD+ en NADH d.m.v. UV/Vis opname, sowel as die skeiding en kwantifisering van 13 glikolitiese spesies afgelei van glukose, insluited ACA en ETOH, saam met GLY en G3P d.m.v. die gebruik en opsporing van 14C- radioaktiewe etikettering. Die nuwe HPLC tegniek was gebruik vir die validering van die kinetiese model wat glikoliese in C2C12 skelet spierselle beskryf, deurom model simulasies met ekserimentele data te vergelyk. 3 inkubasies van C2C12 skelet spierveselekstrak onder verskeie konsentrasies van GLC, FBP, ATP en NAD+ was d.m.v die HPLC tegniek geanaliseer en vergelyk met die ooreenstemende model simulasies. `n goeie ooreensteming tussen eksperimentele data en model simulasies was bevind en valideer die glikolities model van C2C12 skelet spierveselekstrak. Metaboliese kontrole analise van die gevalideerde model was onderneem en dui tot `n meerderheids fluksie kontrolle wat deur die aanvraag van ATP, beskryf deur die ATPase reaksie (99.4%) gehandhaaf word. Dit is in ooreenstemming met wat in die literatuur aangedui is.
AFRIKAANSE OPSOMMING: Diabetes is `n term wat gebruik word om `n groep metaboliese siektes, gekenmerk deur hoë bloed glukose vlakke te beskryf. Regulasie van bloed glukose vlakke word gehandhaaf deur die werking van insulien op teiken weefsels, soos spierweefsel en vetselle. `n Afname in the effektiewe werking van insulin op teikenweefsels lei tot `n wye verskeidenheid van simptome, insluitend skade of versaking van organe soos die oë, niere, senuwees en die hart en lei tot die ontwikkeling van tipe 2 diabetes. Die regulasie van bloed glukose homeostase word oorwegend deur die metaboliese aktiwiteit van skelet spierweefsel (70 – 80%) genhandhaaf. Daarom is daar groot belang in die ondersoek van die versaking van insulin werking, afkomstig van insulien weerstandbediendheid in skelet spierweefsel. In hierdie tesis lê ons die biochemiese karakterisering van die glikolitiese ensieme in C2C12 skelet spiervesels voor. Die kinetiese tempovergelykings, wat die dinamiese gedrag van elke ensiem beskryf, was in `n kinetiese model saamgestel. Die ontwikkeling van `n nuwe HPLC analise tegniek vir die skeiding en kwantifisering van glikolitiese intermediate en kofaktore in selekstrak word ook in hierdie tesis voorgelê. Dié HPLC tegniek handhaaf die skeiding en kwantifisering van die glikolitiese kofaktore; ATP, ADP, AMP, NAD+ en NADH d.m.v. UV/Vis opname, sowel as die skeiding en kwantifisering van 13 glikolitiese spesies afgelei van glukose, insluited ACA en ETOH, saam met GLY en G3P d.m.v. die gebruik en opsporing van 14C- radioaktiewe etikettering. Die nuwe HPLC tegniek was gebruik vir die validering van die kinetiese model wat glikoliese in C2C12 skelet spierselle beskryf, deurom model simulasies met ekserimentele data te vergelyk. 3 inkubasies van C2C12 skelet spierveselekstrak onder verskeie konsentrasies van GLC, FBP, ATP en NAD+ was d.m.v die HPLC tegniek geanaliseer en vergelyk met die ooreenstemende model simulasies. `n goeie ooreensteming tussen eksperimentele data en model simulasies was bevind en valideer die glikolities model van C2C12 skelet spierveselekstrak. Metaboliese kontrole analise van die gevalideerde model was onderneem en dui tot `n meerderheids fluksie kontrolle wat deur die aanvraag van ATP, beskryf deur die ATPase reaksie (99.4%) gehandhaaf word. Dit is in ooreenstemming met wat in die literatuur aangedui is.
Description
Thesis (PhD)--Stellenbosch University, 2020.
Keywords
Skeletal muscle cells, Hyperglycemia, Enzymes -- Analysis, Mathematical model, Diabetes --Genetic aspects, Glycolysis, UCTD