Abstract
Type 2 diabetes mellitus (T2DM) remains a leading culprit in terms of disease burden, afflicting 537 million worldwide and 300,000 individuals in Aotearoa alone, with a disproportionate impact on our Māori, Pasifika, and South Asian populations due to genetic predisposition and inequities in access to healthcare. The global shift towards a Western diet and sedentary living has manifested in a rapidly increasing incidence of diabetes mellitus, which is projected to place further strain on New Zealand’s healthcare system. A hallmark characteristic of T2DM is decreased insulin secretion, resulting in chronic hyperglycaemia. This has long been attributed to a loss of pancreatic β-cell mass. However, a novel theory emerging from recent studies suggests that β-cells possess plasticity and can transdifferentiate into glucagon-producing α-cells which act to raise blood glucose, potentially contributing to the homeostatic dysregulation of glucose that is typical in T2DM.
Forkhead box O protein 1 (FoxO1) is a transcription factor that plays a key role in regulating β-cell differentiation. FoxO1 inhibits β-cell transcription factors, which is thought to lift the suppression of α-cell transcription factors, encouraging α-cell proliferation and β-to-α-cell transdifferentiation. Under healthy conditions, insulin phosphorylates FoxO1 through the Ak strain transforming (Akt) pathway and deactivates it through cytoplasmic translocation. However, under starvation and low energy conditions, AMP-activated protein kinase (AMPK) is theorised to act in contention with Akt by phosphorylating and activating FoxO1 through nuclear translocation, potentially driving β-to-α-cell transdifferentiation.
Uric acid (UA), a pro-inflammatory molecule and product of purine metabolism, is known to act through the AMPK pathway. Furthermore, patients with high serum UA or hyperuricemia have been found to develop T2DM at significantly higher rates than those with normal serum UA. The aim of this project was to determine if hyperuricemia and UA-induced inflammation have an impact on FoxO1 mediation of β-cell plasticity.
Mouse insulinoma (MIN6-CB4) cells were exposed to 0μM (control), 50μM (normal), and 300μM (hyperuricemic levels) of UA, lipopolysaccharide (LPS) (inflammatory control), and 300μM of UA + LPS (combined impact of hyperuricemia and inflammation) and a starvation condition.
Analyses through immunocytochemistry revealed that approximately 70% of FoxO1 was located in the nucleus while 30% was located in the cytoplasm, and this did not significantly change between conditions. Western blot analyses showed no significant change in the expression of Akt-Ser473, an insulin phosphorylation site between conditions. The change in expression of FoxO1-Ser256, an Akt phosphorylation site was unable to be determined due to lack of statistical power. RT-qPCR analyses determined that there was no statistically significant difference in the mRNA expression of FoxO1, β-cell transcription factors such as Pax4, Pdx1, and MafA, the α-cell transcription factor MafB or the inflammatory regulator NFκB between conditions. There was a significant decrease in insulin mRNA expression in the 300μM UA, 300μM UA + LPS, and LPS conditions, and a significant increase in glucagon mRNA expression in the starvation condition. The downregulation of insulin expression supports the pre-existing link between hyperuricemia, inflammation and T2DM. Further investigation could be undertaken into the Akt and AMPK phosphorylation sites on FoxO1 to gain a more comprehensive understanding of FoxO1 activity under hyperuricemic conditions however findings from this project suggest that hyperuricemia may not modulate the AMPK-driven phosphorylation and subcellular localisation of FoxO1 or incite any subsequent changes in β-cell plasticity in MIN6-CB4 cells.