Abstract
Nicotinamide phosphoribosyltransferase (NAMPT) is an enzyme that exists in two forms: intracellular NAMPT (iNAMPT) and extracellular (eNAMPT). The activity of iNAMPT is critical to the intracellular biosynthesis of nicotinamide adenine dinucleotide (NAD+), a substrate that regulates many physiological processes within cells. iNAMPT can be secreted to form eNAMPT, which is less well characterised; with both NAD+ biosynthetic and NAD+-independent cytokine-like properties reported.Several studies that have suggested a key role of iNAMPT activity altering multiple inflammatory processes. These processes include pro-inflammatory cytokine-mediated death and dysfunction, as well as the activation of cytotoxic immune cells. Many of these pathways are associated with pancreatic β-cell destruction, as observed in type 1 diabetes mellitus (T1DM) pathogenesis, however the role of iNAMPT and NAD+ has not been investigated in this context. Moreover, whilst modulating iNAMPT activity has been examined as a therapeutic approach for other autoimmune diseases, its therapeutic potential has yet to be examined in T1DM pathogenesis and development.
The hypothesis of this project is that iNAMPT plays an immunoregulatory role in T1DM pathogenesis, via impacting cytokine signal transduction pathways and immune cell activation. Secondly, we hypothesised that targeting iNAMPT is an attractive therapeutic strategy for the treatment of T1DM.
To investigate this hypothesis, we adopted two main approaches:
1. Using small molecule iNAMPT inhibitors (termed C17 or FK866) and activators (P7C3 or SBI) to determine if iNAMPT plays a role in cytokine-mediated β-cell survival, function, and transcriptome changes in vitro. Cytokine-mediated apoptosis, necrosis, dysfunction, and differential gene expression were examined in isolated pancreatic islets treated for 24 hours with pro-inflammatory cytokines +/- an iNAMPT modulator.
2. Using small molecule iNAMPT inhibitors to determine whether targeting iNAMPT can treat T1DM using an in vivo mouse model of diabetes. The streptozotocin (STZ)-induced T1DM mouse model was treated with an iNAMPT inhibitor daily. Ambient blood glucose and glucose tolerance tests were conducted to assess diabetes development. Intra-islet immune cell infiltration (termed insulitis) and cytokine production levels were measured to determine whether iNAMPT activity impacts inflammatory processes observed in T1DM development.
In vitro experiments revealed that both inhibiting and enhancing iNAMPT protects against cytokine-mediated β-cell death and dysfunction to varying degrees. Transcriptome analysis showed only enhancing iNAMPT with P7C3 blocked deleterious cytokine-mediated gene expression changes but also promoted the cytokine-induced downregulation β-cell enriched genes. The in vivo investigation demonstrated that pharmacologically inhibiting iNAMPT with FK866 in STZ-induced diabetic mice lessened the development of diabetes, although the improvements were mild. Additionally, iNAMPT inhibition partially reduced intra-islet cytokine levels, but did not attenuate insulitis.
In conclusion, this data demonstrates that iNAMPT has some immunomodulatory roles in processes that lead to islet inflammation, in an in vitro and in vivo setting. However, as iNAMPT is reported to play key roles in maintaining normal β-cell function and survival, modulating its activity will have direct effects on β-cell physiology. This is likely why beneficial effects were observed when iNAMPT was activated and inhibited in islets in vitro, and why inhibiting iNAMPT only partially prevented diabetes development in a T1DM mouse model in vivo. Therefore, iNAMPT is not a robust therapeutic target for the treatment of T1DM.
| Date of Award | 1 Nov 2021 |
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| Original language | English |
| Awarding Institution |
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| Supervisor | Paul Caton (Supervisor) & Shanta Persaud (Supervisor) |