Abstract
Diabetes mellitus (DM) is a chronic health condition characterised by high blood glucose concentrations (hyperglycaemia), insufficient insulin secretion from the pancreas and peripheral insulin resistance in most cases. DM can be broadly categorised into two types. Type 1 DM (T1DM) is the least prevalent of the two and results from the autoimmune destruction of insulin producing and secreting β-cells of the pancreas. Type 2 DM (T2DM) on the other hand is acquired through a combination of genetic, environmental and lifestyle factors. T2DM is generally considered to begin with insulin resistance and mild hyperglycaemia, followed by severe hyperglycaemia and loss of β-cell function, and mass, in pancreatic islets. Some research also indicates an increase in the glucagon secreting α-cell mass in T2DM.
The loss of β-cell mass was long thought to be due to apoptosis of the β-cells, however more recent research has indicated that the processes of de-differentiation, where cells lose their mature phenotype, and trans-differentiation, where cells transition from one mature phenotype to another, play key roles in the loss of β-cell function and mass in the development of T2DM. PAX4, PDX1, ARX and FOXO1 are key transcription factors in the determination of islet cell identity with PAX4 and PDX1 promoting β-cell identity and ARX promoting α-cell identity. FOXO1 is a positive regulator of ARX and a negative regulator of PDX1.
High serum uric acid concentrations (hyperuricemia) have been linked to the development of T2DM. Evidence from previous studies suggests that hyperuricemic conditions can interfere with the Transforming Growth Factor-β (TGF-β) and PI3K/Akt signalling pathways which are important pathways regulating these transcription factors. Hyperuricemia can also lead to inflammation, most notably in gout. Inflammation is also strongly associated with the development of T2DM.
It was hypothesised that hyperuricemia in T2DM may contribute to the loss of β-cell mass through de- and trans-differentiation by interfering with the TGF-β and PI3K/Akt signalling pathways, altering expression of PAX4 and ARX. Previous studies in murine (MIN6) and human (1.1B4) cell lines indicate that hyperuricemia induces significant downregulation of PAX4 expression and significant upregulation of ARX transcription.
This study was designed to build upon these previous studies by optimising a protocol to isolate, culture and condition pancreatic islets from mice and assess the effects of hyperuricemia and inflammation in a model closer to the in vivo situation. Three protocols for the collagenase digestion of murine pancreas to extract and isolate islets were tested. Perfusion of the pancreas with collagenase solution via the common bile duct was unsuccessful. Perfusing the pancreas with collagenase solution by injecting collagenase directly throughout the pancreas was unsuccessful. Finally, mincing the pancreas and suspending it in a collagenase solution for digestion proved to be both simple and effective following some optimisation. Culturing of islets post-isolation still requires further optimisation, but preliminary western blot analysis of samples indicates that the method for culturing is viable.