Poster Session 1 - E5
1Andrew Biancolin, 1Emilia Mitova, 1,2Patricia Brubaker
__1 Department of Physiology, University of Toronto; 2 Department of Medicine, University of Toronto; __
Considering that a misalignment of circadian rhythms has been associated with obesity and type 2 diabetes, further exploration of the link between the molecular clock and metabolism is required. Due to its circadian expression in rats and role in positively regulating glucose-dependent insulin secretion, glucagon-like peptide-1 (GLP- 1) is a prime candidate for investigation. This intestinally secreted hormone is released from the L cell and acts as a link between the gut and the pancreas by stimulating insulin secretion in response to nutrient ingestion. Consequently, GLP-1 receptor agonists have been approved for the treatment of type 2 diabetes and have also been shown to have weight loss effects in nondiabetic, obese individuals.
The exact mechanisms coordinating the circadian expression of GLP-1 have yet to be fully elucidated. Known regulators of GLP-1 secretion from the L cell include the Soluble NSF Attachment Protein Receptor (SNARE) proteins, which are involved in regulating the fusion of GLP-1 granules with the membrane. Unpublished mass spectrometry data from our laboratory demonstrates that the SNARE regulatory protein secretagogin exhibits rhythmic expression, as it is highly expressed when GLP-1 secretion peaks and is lowly expressed when GLP-1 secretion troughs, suggesting that secretagogin may also exhibit circadian expression. Additionally, there is unpublished evidence showing that secretagogin-knockout STC-1 cells exhibit decreased GLP-1 secretion. Based on these facts, we hypothesized that circadian expression of secretagogin positively regulates GLP-1 secretion.
The presence of secretagogin has never been shown in the L cell and to demonstrate its expression, we used immunohistochemistry which indicated that it is highly expressed in GLP-1 positive cells from murine intestinal sections. mGLUTag cells were chosen as a cell model for this experiment as they are an adequate representation of L cell physiology and respond to known secretagogues. To test our cell model, mGLUTag cells were stimulated with forskolin in combination with IBMX which significantly increased GLP-1 secretion when compared to control (p<0.001). To further validate our cell model, we used immunocytochemistry to show that the mGLUTag cells express secretagogin. The fluorescent images show that the protein is expressed abundantly throughout the cytoplasm.
To test our hypothesis, RNA was extracted every 4 hours over a 48-hour time course in mGLUTag cells and secretagogin expression was quantified using QPCR (n= 8). The results show that secretagogin exhibits a significant circadian rhythm when analyzed by JTK cycle (p<0.01), with a period of 28 hours and a peak at the 2-hour time point. Rhythmic expression of secretagogin was accompanied by significant circadian rhythms in the core clock genes BMAL1 and PER2 (p<0.01), with periods of 24 and 28 hours respectively. Peak expression of BMAL1 was observed at 10 hours, with a peak in PER2 12 hours later at 22 hours. The antiphasic relationship between BMAL1 and PER2 confirms that the cells were in synchronization. To confirm that the changes in mRNA expression represent the changes in protein levels, western blots were run using protein from the same experiment. A preliminary protein analysis indicates that secretagogin protein levels appeared to peak at 8 and 28 hours, closely following that of BMAL1 (n=2).
In future studies, to determine if this rhythm in secretagogin regulates GLP-1 secretion, siRNA-mediated secretagogin knockdown will be conducted. To verify that the observed effects in vitro are representative of the animal’s physiology, glucose tolerance tests will be conducted in vivo using inducible Cre-lox secretagogin L cell knockout mice. Plasma levels of GLP-1, insulin and glucose will be measured to determine how the absence of L cell secretagogin effects glucose metabolism. Further investigation into the circadian expression of SNARE proteins and their effect on GLP-1 secretion could have implications for understanding the relationship between circadian rhythm misalignment and metabolism.