Examining the transcription factor FOXO1 and its regulation by glucocorticoids and inflammatory mediators in hepatic insulin signalling

Visser, Ankia (2021-12)

Thesis (MSc)--Stellenbosch University, 2021.

Thesis

ENGLISH ABSTRACT: Insulin resistance is the decrease in responsiveness to normal circulating insulin levels, resulting in an attenuated biological response in target tissues (including the liver, muscle, and adipose tissue). In the liver specifically, insulin resistance is no longer able to halt gluconeogenesis, the process whereby glucose production is increased. The forkhead box 1 (FOXO1) transcription factor, the protein responsible for the transcription of enzymes required for gluconeogenesis, remains active in the nucleus. There are many factors that can contribute to the development of insulin resistance, including stress and inflammation. In chronic stress, the hypothalamic- pituitary-adrenal (HPA) axis becomes hyperactive resulting in elevated glucocorticoid (GC) levels such as cortisol (F), whereas chronic inflammation is associated with an increase in the production and secretion of pro-inflammatory cytokines such as tumour necrosis factor α (TNF-α) and interleukin 6 (IL-6). Both GCs and elevated pro- inflammatory cytokine levels have been implicated in the development of insulin resistance, however, stress and inflammation-induced hepatic insulin resistance is not well understood, particularly the effects stress and inflammation have on the regulation of FOXO1. For this reason, the main aim of this study was to investigate the effects GCs and pro-inflammatory cytokines have on FOXO1 expression and activity in the liver cell line HepG2. The results showed that GCs, as well as pro-inflammatory cytokines impair insulin signalling in liver cells, although to different extents and time of exposure. More specifically, Dexamethasone (Dex) increased FOXO1 mRNA and protein expression and had the most pronounced activity on the promoter of FOXO1, whilst F showed activity on the promoter of FOXO1 but only increased FOXO1 protein expression. Both pro-inflammatory cytokines increased FOXO1 mRNA expression but had no effect on the promoter of FOXO1, and only TNF-α increased FOXO1 protein levels. In addition, TNF-α appeared to act co-operatively with both GCs, increasing FOXO1 mRNA expression, whereas IL-6 inhibited GC-induced effects on FOXO1. Finally, both GCs and pro-inflammatory cytokines prevented insulin from inhibiting FOXO1 activity. To the best of our knowledge, this is the first study to investigate stress- and inflammation-induced insulin resistance on FOXO1 both alone and in combination. The findings of this study suggest FOXO1 dysregulation as being a potential contributor to hepatic insulin resistance induced by stress and inflammation.

AFRIKAANSE OPSOMMING: Insulienweerstandigheid is die afname in reaksie op normale insulienvlakke in sirkulasie, wat lei tot ʼn verswakte biologiese reaksie in teikenweefsels (insluitend die lewer-, spier- en vetweefsel). In die geval van die lewer, kan insulienweerstandigheid nie meer glukoneogenese, die proses waardeur die produksie van glukose verhoog word, stop nie. Die transkripsiefaktor van die vurkkas 1 (FOXO1), die proteïen wat verantwoordelik is vir die transkripsie van ensieme wat benodig word vir glukoneogenese, bly aktief in die kern. Daar is vele faktore wat kan bydra tot die ontwikkeling van insulienweerstandigheid, insluitend spanning en inflammasie. In chroniese stres word die hipotalamus-hipofise-bynier (HPA) hiperaktief, wat lei tot verhoogde glukokortikoïedvlakke (GC vlakke) soos kortisol (F), terwyl chroniese inflammasie geassosieer word met ʼn toename in die produksie en afskeiding van pro- inflammatoriese sitokiene soos as tumornekrose faktor α (TNF-α) en interleukien 6 (IL- 6). Beide GC’s en verhoogde pro-inflammatoriese sitokienvlakke word geïmpliseer in die ontwikkeling van insulienweerstandigheid, maar spanning en inflammasie- geïnduseerde lewerinsulienweerstandigheid word nog nie goed verstaan nie, veral die uitwerking wat spanning en inflammasie op die regulering van FOXO1 het. Om hierdie rede was die hoofdoel van hierdie studie om die effek wat GC's en pro-inflammatoriese sitokiene op FOXO1-uitdrukking en -aktiwiteit in die lewersellyn HepG2 het, te ondersoek. Die resultate toon dat GC’s, sowel as pro-inflammatoriese sitokiene, insulienseining in lewerselle benadeel, hoewel in verskillende mate en blootstellingstyd. Meer spesifiek het Dexamethasone (Dex) die FOXO1-mRNA en proteïenuitdrukking verhoog en het die mees beduidende aktiwiteit op die promotor van FOXO1 gehad, terwyl F aktiwiteit getoon het op die promotor van FOXO1, maar slegs die FOXO1-proteïenuitdrukking verhoog het. Beide pro-inflammatoriese sitokiene het FOXO1 mRNA-uitdrukking verhoog, maar het geen effek op die promotor van FOXO1 gehad nie, en slegs TNF-α het FOXO1-proteïenvlakke verhoog. Daarbenewens blyk dit dat TNF-α saamwerk met albei GC’s, wat die uitdrukking van FOXO1 mRNA verhoog, terwyl IL-6 GC-geïnduseerde uitwerkinge op FOXO1 inhibeer. Ten slotte het beide GC's en pro-inflammatoriese sitokiene verhinder dat insulien FOXO1-aktiwiteit inhibeer. Na die beste van ons wete, is dit die eerste studie wat insulienweerstandigheid weens spanning en inflammasie op FOXO1, alleen en in kombinasie, ondersoek. Die bevindings van hierdie studie dui daarop dat FOXO1-disregulering ʼn potensiële bydraer is tot insulienweerstandigheid in die lewer wat deur spanning en inflammasie veroorsaak word.

Please refer to this item in SUNScholar by using the following persistent URL: http://hdl.handle.net/10019.1/123645
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