Focus: Lessons learned on diabetes pathogenesis based on human pancreas/islets

Title:

Designing a cell culture model to study the effects of ER stress involved in the pathogenesis of type 2 diabetes.

Background:

Diabetes is a debilitating disease that afflicts millions of individuals worldwide, and the statistics rise each year. Diabetes is associated with various ailments which reduce the quality of life, and has been shown to be associated with seemingly unrelated diseases or conditions. Finding an effective treatment for diabetes requires efficacious treatments that could be studied and screened in a model that closely resembles the pathogenesis of the disease.

Aims:

An INS-1E cell line model was investigated to study ER stress in the development of type 2 diabetes, with the potential to evaluate the efficacy of treatments associated with both ER stress and diabetes simultaneously.

Methods:

Pancreatic beta-cells (INS-1E) were treated with high glucose (33.3 mM) media with the natural ER stress inducer (interleukin-1beta) or synthetic compounds known to induce ER stress (tunicamycin, brefeldin-A, and thapsigargin) to evaluate the development of ER stress and its effect on insulin production. The MTT assay was used to assess the effect of the high glucose and ER stress inducers on cell viability, and cell electrical impedance using the Roche xCelligence system was used as a secondary cell viability assay. Nitric oxide was quantified to provide an indirect link between glucotoxicity and ER stress in the natural model involving the elevated interleukin-1beta levels. Flow cytometric analysis using Annexin-V and propidium iodide was used to validate the time points used for the model. An insulin ELISA was performed to determine the effect of hyperglycaemia and ER stress on the cells ability to produce and secrete insulin.

Results:

Data obtained from MTT, cell electrical impedance, and flow cytometric analysis of high glucose indicated that high glucose appeared to confer an initial protective feature to the cells, which later became deleterious to the cells as the high glucose became chronic. The ER stress inducers showed a gradual decline in cell viability over time; however, the synthetic ER stress inducers appeared to display far more potent cytotoxicity effects on the pancreatic beta cells than interleukin-1beta. Nitric oxide was only present when the cells were treated with interleukin-1beta, with high glucose exacerbating the production of nitric oxide. Insulin levels were markedly decreased with hyperglycaemia and the synthetic ER stress inducers, but retained normal levels when cultured with interleukin-1beta.

Discussion:

While each of the assays described give similar categories of results (i.e. cell viability), they address different aspects involved in biological systems, and each have their own advantages and disadvantages. The MTT data gave insight into cellular metabolism that is important to consider when studying metabolic diseases such as diabetes; however, the assay is an endpoint assay. The cell electrical impedance assay addresses this by providing real-time data of cells attaching and detaching from the surface of the plate, providing both cell viability and cell proliferation data, but lacking additional biochemical information such as pathways activated or modes of potential cell death. The flow cytometric analysis provides much needed data regarding the mode of cell death cells may experience in the presence of high glucose and/or ER stress inducers, as well as size dimension variations of the cells, but is again an endpoint assay that lacks real-time factors. Nitric oxide quantification gives an indication of potential inflammation within the model, which is a pivotal aspect to be considered when studying diabetes and ER stress. The insulin ELISA data answered the vital diabetes-related question of how the model, or any treatments screened in the model, influenced the production and secretion of insulin, the absence of which is often indicative of diabetes. Using these methods to build the foundation of an optimal cell culture model to study diabetes and various related conditions associated with diabetes and ER stress, the researcher is able investigate more specific biochemical pathways and processes much quicker and more efficiently.

Designing a cell line model to study the effects of endoplasmic reticulum stress involved in the progression of diabetes