The regulatory design of glycogen metabolism in mammalian skeletal muscle

Palm, Daniel Christiaan (2013-03)

Thesis (PhD)--Stellenbosch University, 2013.

Thesis

ENGLISH ABSTRACT: It is widely accepted that insufficient insulin-stimulated activation of muscle glycogen synthesis is one of the major components of non-insulin-dependent (type 2) diabetes mellitus. Glycogen synthase, a key enzyme in glycogen synthesis, is extensively regulated, both allosterically (by glucose-6-phosphate, ATP, and other ligands) and covalently (by phosphorylation). Although glycogen synthase has been a topic of intense study for more than 50 years, its kinetic characterization has been confounded by its large number of phosphorylation states. Questions remain regarding the function of glycogen synthase regulation and the relative importance of allosteric and covalent modification in fulfilling this function. The regulation of glycogen synthase and glycogen phosphorylase, the enzyme that catalyses the degradation of glycogen chains, are reciprocal in many respects. In the present research, using mathematical modelling, we aim to establish the function of the allosteric and covalent regulation of glycogen synthase and glycogen phosphorylase in muscle and, in the case of glycogen synthase, the relative importance of these two mechanisms in performing this function. In order to realize these aims it is essential that a detailed kinetic model of glycogen metabolism is constructed. We begin with a thorough review of the kinetics and regulation of glycogen synthase inwhich we propose that both allosteric and covalent modification of glycogen synthase can be described by a Monod-Wyman-Changeux model in terms of apparent changes to L0, the equilibrium constant between the T and R conformers. We then proceed to develop a rate equation according to the proposed Monod-Wyman-Changeux model and determine values for its kinetic parameters from published experimental data using non-linear least-squares regression. We show that the application of the Monod-Wyman-Changeux model to glycogen synthase kinetics also has important implications for the rate equations of enzymes that catalyse the phosphorylation and dephosphorylation of glycogen synthase. We formalize these implications for a generic protein that follows Monod-Wyman-Changeux-type conformational change and then also show how the findings apply to glycogen synthase. Taking into account the kinetic model of glycogen synthase and how it also influences the covalent regulation of the enzyme, we proceed to construct a detailed mathematical model of glycogen synthesis that includes the glycogen synthase phosphorylation cascade. A variation of this model in which glycogen synthase phosphorylation is described with a single parameter is also provided. We reuse an existing model of muscle glycogenolysis and also combine these models in an overall model of glycogen metabolism. Finally, we employ the theoretical frameworks of metabolic control analysis, supply-demand analysis, and co-response analysis to investigate the function of glycogen synthase and glycogen phosphorylase regulation. We show that the function of glycogen synthase regulation is not flux control, as assumed in the textbook view, but rather the maintenance of glucose-6-phosphate within a narrow range far from equilibrium. Similarly, we show that regulation of glycogen phosphorylase functions to minimize variation in cellular energy charge in the face of highly variable energy demand. We conclude with an appeal for a renewed interest in the enzyme kinetics of muscle glycogen metabolism.

AFRIKAANSE OPSOMMING: Daar word wyd aanvaar dat onvoldoende insulien-gestimuleerde aktivering van spierglikogeensintese een van die hoofkomponente van insulien-onafhanklike (tipe 2) diabetes mellitus is. Glikogeensintase, ’n sleutelensiem in glikogeensintese is onderworpe aan breedvoerige regulering, beide allosteries (deur glukose-6-fosfaat, ATP, en ander ligande) en kovalent (deur fosforilering). Alhoewel glikogeensintase reeds vir meer as 50 jaar deeglik bestudeer word, word die kinetiese karakterisering daarvan bemoeilik deur die groot aantal fosforilasiestate waarin die ensiem voorkom. Daar is steeds vrae betreffende die funksie van die regulering van glikogeensintase en die relatiewe bydrae van allosteriese en kovalente regulering in die vervulling van hierdie funksie. Die regulering van glikogeensintase en glikogeenfosforilase, die ensiem wat die afbraak van glikogeenkettings kataliseer, is in baie opsigte resiprook. In hierdie studie beoog ons om met die hulp van wiskundige modellering vas te stel watter funksie die regulering van glikogeensintase en glikogeenfosforilase vervul en, in die geval van glikogeensintase, wat die relatiewe belang is van allosteriese en kovalente regulering in die vervulling van hierdie funksie. Om hierdie oogmerke te verwesentlik is dit nodig dat ’n kinetiese model van glikogeenmetabolisme ontwikkel word. Ons begin met ’n omvattende oorsig van die kinetika en regulering van glikogeensintase waarin ons voorstel dat beide die allosteriese en kovalente regulering van glikogeensintase beskryf kan word met die Monod-Wyman-Changeux model in terme van oënskynlike veranderings aan L0, die ekwilibriumkonstante tussen die T en R konformasies. Ons gaan dan voort om ’n snelheidsvergelyking te ontwikkel volgens die voorgestelde Monod-Wyman-Changuex-model en bepaal ook die waardes van hierdie vergelyking se parameters vanaf gepubliseerde eksperimentele data deur middel van nie-lineêre kleinste-vierkantsregressie. Ons wys dat die toepassing van die Monod-Wyman-Changuex-model op glikogeensintase-kinetika belangrike gevolge het vir die snelheidsvergelykings van die ensieme wat die fosforilering en defosforilering van glikogeensintase kataliseer. Ons formaliseer hierdie gevolge vir ’n generiese Monod-Wyman-Changeux-tipe proteïen en wys dan ook hoe die bevindings op glikogeensintase van toepassing is. Met inagneming van die kinetiese model vir glikogeensintase en hoe dit die kovalente regulering van die ensiem be¨ınvloed, gaan ons voort om ’n gedetaileerde wiskundige model van glikogeensintese, wat ook die glikogeensintase-fosforileringskaskade insluit, te ontwikkel. ’n Variasie op hierdie model waarin die fosforilering van glikogeensintase deur ’n enkele parameter beskryf word, word ook voorsien. Ons herbruik ’n bestaande model van spierglikogenolise en kombineer ook hierdie modelle in ’n oorkoepelende model van glikogeenmetabolisme. Uiteindelik span ons die teoretiese raamwerke van metaboliese kontrole-analise, vraag-aanbod-analise, en ko-responsanalise in om die funksie van die regulering van glikogeensintase en glikogeenfosforilase te ondersoek. Ons wys dat die funksie van die regulering van glikogeensintase nie fluksiekontrole, soos algemeen in handboeke aangeneem word, is nie, maar liewer dat dit glukose-6-fosfaat handhaaf binne ’n noue band ver vanaf ekwilibrium. Insgelyks wys ons dat die regulering van glikogeenfosforilase funksioneer om variasie in sellulˆere energielading te beperk ten spyte van hoogs wisselende vlakke van energie-aanvraag. Ons sluit af met ’n pleidooi vir hernieude belangstelling in die ensiemkinetika van glikogeenmetabolisme in die spier.

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