Novel targets for the development of drugs for Type 2 Diabetes Mellitus

Type 2 Diabetes Mellitus (T2DM) is a modern metabolic disorder. T2DM is related with modern lifestyle and results when the body is unable to produce sufficient insulin or respond properly to insulin, which is the hormone that regulates the levels of glucose in blood. The aim of this project was to define novel targets for the possible development of drugs for T2DM. In the thesis we developed 3 models that can assist the screening of novel drugs for T2DM.  .

In the first model that is presented in this thesis it is shown that resistin downregulation in mouse adipocytes would provide an alternative for PPARγ agonist activity, and assay frequently used in the past to define novel drugs for T2DM. The results revealed a significant correlation between the EC50 for PPARγ activation in a reporter gene cell line and the IC50 for resistin downregulation in adipocytes. The outcomes indicate that PPARγ mediated downregulation of resistin might promote insulin sensitivity and that resistin can be used as a target for searching insulin sensitizing compounds.

In the second model presented in this thesis, the detection of TLR-4 mediated gene expression was investigated as a possible bioassay to detect candidate antidiabetic drugs able to inhibit resistin mediated TLR-4 activation and subsequent effects. Inhibition of LPS mediated TLR-4 signaling by two known TLR-4 inhibitors could be demonstrated as well as the inhibition of resistin mediated TLR-4 signaling by these two known TRL-4 inhibitors. It was concluded that the TLR-4 reporter gene cell line provides a novel tool for detection of resistin antagonists and might be of use as a new target to screen for a possible new type of antidiabetic drugs.

A third in vitro model that is presented in this thesis for the search on novel targets for T2DM drugs consists of a model that mimics inflamed adipocytes. Adipocytes (3T3-L1) were co-cultured directly with activated macrophages (RAW264.7) producing an environment for testing compounds under conditions more similar to the in vivo situation in T2DM patients. A comparison of our results to a study on human visceral fat of obese non-diabetic and obese diabetic patients indicated that AUH, NAGK, pCYT2, NNMT, STK39 and CSNK2A2 genes and proteins have the potential of being prognostic biomarkers.

To further increase the possibility for identification of novel targets for antidiabetic drugs via the possible role of resistin, the mechanism behind resistin mediated effects was investigated. Human adipocytes were exposed to resistin and the effects on gene expression were analysed by RNA sequencing. Based on these observations it was proposed that resistin dependent gene expression is related to modifications in cellular maintenance processes, slowing down metabolic changes resulting in energy conservation. Under these circumstances of metabolic adaptation in the presence of resistin, excess food consumption as in modern societies can result in obesity, inflammation and insulin resistance.

Altogether, there is a sincere hope that the models presented in this thesis will contribute to the definition of novel therapeutic strategies and enable answering some of the questions about a complex disease such as T2DM that has been studied for so long time.