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Investigating the influence of insulin on autophagy induction and autophagic turnover in mycobacterium tuberculosis-infected macrophages
Thesis (MSc)--Stellenbosch University, 2022.
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| Format: | Thesis |
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Stellenbosch : Stellenbosch University
2022
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| _version_ | 1867613763580985344 |
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| access_status_str | Open Access |
| author | Scholtz, Denise |
| author2 | Kinnear, Craig J. |
| author_browse | Kinnear, Craig J. Scholtz, Denise |
| author_facet | Kinnear, Craig J. Scholtz, Denise |
| author_sort | Scholtz, Denise |
| collection | Thesis |
| dc_rights_str_mv | Stellenbosch University |
| description | Thesis (MSc)--Stellenbosch University, 2022. |
| format | Thesis |
| id | oai:scholar.sun.ac.za:10019.1/125037 |
| institution | Stellenbosch University (South Africa) |
| language | en_ZA |
| last_indexed | 2026-06-10T12:41:19.170Z |
| license_str | Other — see source repository |
| provenance_str_mv | Harvested via OAI-PMH from SUNScholar — Stellenbosch University Repository |
| publishDate | 2022 |
| publishDateRange | 2022 |
| publishDateSort | 2022 |
| publisher | Stellenbosch : Stellenbosch University |
| publisherStr | Stellenbosch : Stellenbosch University |
| record_format | dspace |
| source_str | SUNScholar — Stellenbosch University Repository |
| spelling | oai:scholar.sun.ac.za:10019.1/125037 Investigating the influence of insulin on autophagy induction and autophagic turnover in mycobacterium tuberculosis-infected macrophages Scholtz, Denise Kinnear, Craig J. Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences. Molecular Biology and Human Genetics. Mycobacterium tuberculosis -- Mortality Type 2 diabetes Autophagy Macrophages Thesis (MSc)--Stellenbosch University, 2022. ENGLISH ABSTRACT: Tuberculosis (TB), one of the oldest known human diseases and a leading cause of death worldwide, is caused by the bacterium Mycobacterium tuberculosis. One-quarter of the world’s population is infected with latent M. tuberculosis, and as a result of immunodeficiency or immunocompromising, TB can be reactivated. Type 2 Diabetes Mellitus is associated with an elevated risk of TB-related drug resistance as it could lower exposure to anti-TB drugs, increase treatment failure and the risk of recurrent TB. Due to the increasing prevalence of TB/T2DM comorbidity worldwide, the optimal treatment of TB/T2DM comorbidity is important. The need for host-directed therapy (HDT) is crucial to improve treatment efficiency and modulating the host response could lead to adjunct therapies for the combined treatment of TB and other comorbidities. Autophagy is a fundamentally important process that maintains the cellular homeostasis and survival of the cell and is critical for the regulation of an array of immune responses. It has been linked to the management of both TB and T2DM and therefore provides a strategy for HDT. Preliminary findings on the most commonly used first-line T2DM medication, Metformin, suggests that it may be valuable to control TB by reducing inflammation associated with immune pathology, as well as increasing anti-mycobacterial activities, such as autophagy. This study hypothesised that insulin might have a similar effect as metformin, by inducing autophagy and stimulating autophagic turnover in M. tuberculosis-infected macrophages. As this was a pilot study, we initially focused on Insulin opting to evaluate glybenclamide in future studies. We aimed to do this by investigating the effect of insulin on autophagy induction and turnover in uninfected and M. tuberculosis-infected cells, as well as elucidating the effects of insulin on mycobacterial killing in THP-1 macrophages. The autophagic process was evaluated by treating cultured THP-1 macrophages with insulin, and measured the expression of key autophagic markers, LC3B-II and p62, over time in uninfected and in M. tuberculosis-infected macrophages. The expression of markers was determined through western blotting, and relevant statistical analyses were conducted. Colony forming units (CFUs) were also enumerated to investigate the effect of insulin on mycobacterial replication and clearance. Our results suggested that the treatment of macrophages with a high dosage of insulin (1000 nM) will inhibit autophagy, whereas treatment with 100 nM insulin increased autophagic Stellenbosch University https://scholar.sun.ac.za iv activity in uninfected macrophages. However, when infected with M. tuberculosis, insulin treatment seemed to induce autophagy, but negatively affected the turnover and clearance of protein markers. Additionally, CFU enumeration indicated no apparent differences between insulin treated macrophages and controls in their ability to enhance mycobacterial killing and clearance of bacilli from the cells. To conclude, this study provided valuable information on the effect of insulin treatment on autophagy in the context of M. tuberculosis-infection. More research is needed to fully elucidate the extent of the effects of exogenous insulin as a potential HDT. AFRIKAANSE OPSOMMING: Tuberkulose (TB) is een van die oudste bekende menslike siektes en is 'n leidende oorsaak van sterftes wêreldwyd, wat veroorsaak word deur die bakterieë Mycobacterium tuberculosis. Een kwart van die wêreld is besmet met latente M. tuberculosis, en as gevolg van immuniteitsgebrek of immunokompromisasie kan TB heraktiveer word. In Tipe 2 Diabetes Mellitus (T2DM) is dit die geval, en as gevolg van die hoë voorkoms van diabetes wêreldwyk, is die optimale bestuur van gekombineerde tuberkulose en diabetes belangrik. T2DM word geassosieer met 'n verhoogde risiko van TB-verwante dwelmweerstand, aangesien dit blootstelling aan anti-TB-middels kan verlaag, asook behandelingsmislukking en die risiko van herhalende TB kan verhoog. Die behoefte aan gasheergerigte terapie is van kardinale belang om behandelingsdoeltreffendheid te verbeter en die moderering van die gasheerreaksie kan lei tot byvoeglike terapieë vir die behandeling van TB. Outofagie is 'n fundamenteel belangrike proses wat die sellulêre homeostase en oorlewing van die sel handhaaf en is van kritieke belang vir die regulering van 'n wye verskeidenheid immuunresponse. Dit is gekoppel aan die bestuur van beide TB en T2DM en bied dus 'n potentsiële strategie vir gasheergerigte terapie. Voorlopige bevindinge oor algemeen gebruikte T2DM-medikasie, metformien, dui daarop dat dit voordelig kan wees om TB te beheer deur inflammasie wat verband hou met die immuunpatologie te verminder en anti-mikobakteriële aktiwiteite soos outofagie te verbeter. Hierdie studie het gehipotetiseer dat insulien 'n soortgelyke effek as metformien kan hê, deur outofagie te bevorder en outofagiese omset in M. tuberculosis-besmette makrofage te stimuleer. Ons het daarop gemik om dit te doen deur die effek van insulien op outofagieinduksie en omset in onaangeraakte en besmette selle te ondersoek, asook om die effekte van insulien op mikobakteriese moord in THP-1 makrofage te verlig. Hierdie studie het die outofagiese proses geëvalueer deur gekultiveerde THP-1 makrofage met insulien te behandel, en het mettertyd uitdrukking van kern outofagiese merkers, LC3B-II en p62, toegelig, in ongeïnfekteerde makrofage, asook M. tuberculosis-geïnfekteerde makrofage. Die uitdrukking van merkers is bepaal deur westerse blotting, waarna die resultate met die toepaslike statistiese programme geanaliseer is. Kolonie-formende eenhede (KFE) is ook opgesom om die effek van insulien op mikobakteriese replikasie en opruiming te ondersoek. Stellenbosch University https://scholar.sun.ac.za vi Ons resultate het voorgestel dat die behandeling van 'n hoë dosis insulien (1000nM) outofagie kan inhibeer, terwyl die behandeling met 100nM insulien verhoogde outofagiese aktiwiteit in onaangeraakte makrofage oor die algeheel veroorsaak het. Met die aanslag van infeksie met M. tuberculosis, het insulienbehandeling outofagie geinduseer, maar die omset en opruiming van die proteïenmerkers negatief beïnvloed. Daarbenewens het KFE-opsomming geen oënskynlike verskille tussen insulien behandelde makrofage en kontroles aangedui in hul vermoë om mikobakteriese moord en opruiming van bacilli uit die selle te verbeter nie. Om af te sluit, het hierdie studie waardevolle inligting verskaf oor die effek van insulienbehandeling op outofagie in die konteks van M. tuberculosis-infeksie, ten spyte van meer navorsing wat nodig is om die omvang van die effekte ten volle van eksogene insulin as 'n potensiële gasheergerigte terapie te verduidelik. Masters 2022-02-17T09:25:13Z 2022-04-29T12:51:21Z 2023-03-18T03:00:07Z 2022-02 Thesis http://hdl.handle.net/10019.1/125037 en_ZA Stellenbosch University viii, 103 pages : illustrations application/pdf Stellenbosch : Stellenbosch University |
| spellingShingle | Mycobacterium tuberculosis -- Mortality Type 2 diabetes Autophagy Macrophages Scholtz, Denise Investigating the influence of insulin on autophagy induction and autophagic turnover in mycobacterium tuberculosis-infected macrophages |
| title | Investigating the influence of insulin on autophagy induction and autophagic turnover in mycobacterium tuberculosis-infected macrophages |
| title_full | Investigating the influence of insulin on autophagy induction and autophagic turnover in mycobacterium tuberculosis-infected macrophages |
| title_fullStr | Investigating the influence of insulin on autophagy induction and autophagic turnover in mycobacterium tuberculosis-infected macrophages |
| title_full_unstemmed | Investigating the influence of insulin on autophagy induction and autophagic turnover in mycobacterium tuberculosis-infected macrophages |
| title_short | Investigating the influence of insulin on autophagy induction and autophagic turnover in mycobacterium tuberculosis-infected macrophages |
| title_sort | investigating the influence of insulin on autophagy induction and autophagic turnover in mycobacterium tuberculosis infected macrophages |
| topic | Mycobacterium tuberculosis -- Mortality Type 2 diabetes Autophagy Macrophages |
| url | http://hdl.handle.net/10019.1/125037 |
| work_keys_str_mv | AT scholtzdenise investigatingtheinfluenceofinsulinonautophagyinductionandautophagicturnoverinmycobacteriumtuberculosisinfectedmacrophages |