Högskolan i Skövde

Pancreatic β-cells produce insulin that balances the level of blood glucose. These cells also produce Synaptic-like micro-vesicles (SLMV) in which γ-aminobutyric acid (GABA) is stored; a metabolite known to be effective in the control of pancreatic endocrine functions in human islets of Langerhans. It is demonstrated in the previous studies conducted by researchers that biogenesis of SLMVs is strongly dependent on a group of Adaptor Protein complex known as AP3, and intracellular trafficking will be impaired in absence of AP3. In this study in order to understand the components of SLMVs better and to assess the role of AP3 in SLMV production and secretion, the exocytosis mechanisms of these vesicles were studied in rat pancreatic β-cell INS1 cell line in both wild type and knocked out AP3 cells using CRISPR/Cas9 technology. Cells were also transfected with plasmids expressing proteins related to SLMVs and their fusion site on the plasma membrane. Samples, either fixed or live, were imaged using fluoroscent microscopes. Based on the results, a moderate correlation was found for colocalization of AP3 and SLMVs. Moreover, exocytosis events, before and after glucose stimulation, were imaged by TIRF microscope and the results suggested an increase in fusion events for SLMVs onto the plasma membrane, immediately after glucose addition. We propose here a possible relationship between AP3 and SLMVs biogenesis and secretion which may contribute to future development of an explanatory model if confirmed with larger samples.

Popular scientific summary

In Toronto in 1921, Fredrick Banting a young surgeon and Charles Best a medical student discovered insulin. Two years later they were awarded the Nobel Prize in Physiology or Medicine shared with two other scientists who put great effort into the discovery and purification of insulin. The name stems from the Latin word insula meaning "island" since this hormone is produced and secreted by a cluster of cells known as the islets of Langerhans found in the pancreas. The journey of insulin begins with its synthesis and production, continues with quality control, delivery, and eventually action, which are all exquisitely regulated in several organs or “stations” in the body. Its main function as soon as it is released in the blood, is to uptake glucose and to suppress the glucose production cycle from cells. Commercially produced insulin is generally used in the treatment of diabetes. In people with diabetes, there is either a complete absence of insulin, known as type 1 diabetes, or the process of insulin production is impaired due to diminished insulin secretion and/or insulin resistance which is type 2 diabetes. The islets of Langerhans are mostly comprised of beta cells (β-cells) but also alpha cells (α-cells) δ, γ and ε cells, and it's the loss or dysfunction of insulin-producing β cells in the pancreas that results in deregulation of glucose homeostasis. However, insulin secretion is not the only important function of these cells. Another molecule which is produced and secreted by the same cells is neurotransmitter γ[1]aminobutyric acid (GABA), which has recently been shown to slow down or even reverse the loss of β cells. In order to use the capacity of GABA in treatment of diabetes, an understanding of biogenesis, storage, and secretion of GABA is pivotal. β cells have been studied and demonstrated to produce a type of vesicles named synaptic-like micro-vesicles (SLMVs). Vesicles are structures made of phospholipid layers, in or outside of cells that contain and transport different molecules. There are some similarities between SLMVs and synaptic vesicles that are present in neurons in brain, they both contain GABA, they are abundant, and regulated fusion with the plasma membrane occurs for both types of vesicles. Even though there have been some studies on the SLMVs, there are yet many undiscovered aspects of them that, if known better, may help find a new treatment for diabetes. For instance, in previous studies the role of a protein complex called AP3 is shown to be of high importance regarding the production of these SLMVs, yet not very well known. In this study, with the help of advanced technology and methods such as TIRF microscopy (Total Internal Reflection Fluorescence), β cells were imaged and those images were analyzed in order to understand the role of AP3 in the secretion of SLMVs from these cells. Also, we detected the existence of some molecules close to the site of SLMV secretion as they were suggested in previous studies to have a connection with this type of secretory vesicles. Results from this study suggest that there is a moderate correlation between SLMVs and proteins that are involved in secretion of other vesicles in the pancreatic β cells, and also a possible relationship between AP3 protein complex and SLMV production. The results and information from this study can assist future research projects as well as introducing new potential treatments for diabetes.