Abstract
Polymorphisms in the gene T-cell factor 7-like 2 (TCF7L2) carry one the biggest genetic linkage to the development of type 2 diabetes. However the exact mechanism through which this occurs is still under debate, although these polymorphisms are suspected to be somehow due to increases in TCF7L2 gene expression. The TCF7L2 transcriptional co-factor, β-catenin, is regulated by several metabolic hormones in peripheral tissues and has been shown to be important for the formation and function of neuron synapses. Overexpression of TCF7L2 causes the translocation of β-catenin out of the cytoplasm and into the nucleus, potentially affecting its functions. It is possible that modulated TCF7L2 expression is affecting β-catenin function at the synapse in hypothalamic neurons and contributing to the metabolic dysregulation in individuals with the TCF7L2 polymorphisms. We aim to determine if various feeding related stimuli are able to increase β-catenin stabilisation in hypothalamic nuclei that control glucose homeostasis and if this stabilisation of β-catenin is affecting neuron function.
The stabilisation of β-catenin in fasted versus fed rats was assessed in the hypothalamus through immunohistochemistry. The paraventricular (PVN), dorsomedial (DMH) and arcuate (Arc) hypothalamic neuclei all showed increases in the number of cells stained positive for β-catenin. These regions also showed increased expression of the β-catenin responsive gene markers of Axin2, CyclinD1, Cyclin D2 and c-myc.
To investigate which feeding related stimuli was inducing the changes observed after feeding the hypothalamic neuronal model of GT1-7 cells was used. GT1-7 cells were stimulated with leptin, glucose, insulin, the glucagon-like peptide-1 (GLP-1) analogue Exendin-4 and alpha-melanocyte stimulating hormone (alpha-MSH). Glucose, insulin, Exendin-4 and alpha-MSH all had the capacity to stabilise β-catenin through increasing phosphorylation at the serine 552 and 675 residues on β-catenin. In the case of Exendin-4, this was shown to be a PKA dependent effect.
Glucose and Exendin-4 were administered to rats in an attempt to replicate the observations made in cell culture. Despite glucose not showing an effect on the stablisation of β-catenin, Exendin-4 increased the number of positively stained cell for β-catenin in the PVN, DMH and Arc using immunohistochemistry. This was further supported by elevations in Axin2, CyclinD1, cyclinD2 and c-myc transcripts.
Finally, β-catenin expression was suppressed using siRNA transfection, or stabilised via inhibition of GSK-3β in GT1-7 cells prior to stimulation with Exendin-4. Decreased β-catenin expression reduced the secretary capacity of the cells when stimulated with Exendin-4, while increasing β-catenin stability had the opposite effect. This indicated that β-catenin was important for neurosecretion.
Additionally, there was also a further study that sought to determine if the phosphorylation of Akt in the brain observed after administration of the atypical antipsychotic medication, clozapine, is dependant on a reported side-effect of this class of drug in which insulin is hypersecreted. Activation of Akt by clozapine has been linked to its procognitive effects in schizophrenic patients, This has previously been thought to be a direct drug effect. Rats which were pretreated with octreotide, a somatoestatin analogue, to suppress insulin secretion from β cells before clozapine administration. Octreotide/clozapine treated rats all displayed severely lowered Akt phosphorylation in cognitive brain regions, including the hippocampus, cortex and amygdala, when compared to rats that received clozapine alone.
The results obtained in this thesis outline novel mechanisms in which β-catenin can be stabilised in hypothalamic neurons and indicates the importance that β-catenin has in synaptic function. Various metabolic stimulants are contributing to increases in the stabilisation of β-catenin in hypothalamic nuclei responsible in the regulation of glucose homeostasis. Increasing levels of β-catenin is able to increase the neurosecretary capacity of neurons. Conversely, suppressing β-catenin expression lowers neurosecretion. As TCF7L2 polymorphisms increase the expression of the TCF7L2, it is possible that there is increased interaction between TCF7L2 and β-catenin, sequestering it into the nucleus and lowering localised synaptic levels of β-catenin. This would impair neurosecretion and may be contributing the glucose homeostatic dysregulation in individuals carrying a TCF7L2 polymorphism. Results also showed that the clozapine induced Akt phosphorylation in the brain, which has been linked to positive cognitive outcomes in schizophrenic patients, is dependent on clozapine induced insulin secretion as opposed to a direct drug effect.