The role of salt inducible kinases in the regulation of the mammalian target of rapamycin pathway in adipocytes

English. Salt inducible kinase 2 (SIK2) is found abundantly in adipose tissue and may have a role in the development of diabetes. SIK2 is downregulated in insulin-resistant and obese individuals, thereby providing a new potential target for therapeutic solutions. Dysfunctional adipose tissue is observed in obesity and this is correlated with impaired insulin signalling, which might contribute to imbalanced adipokine secretion and ectopic fat deposition. Degradation and recycling of cellular components are important for the maintenance of the cell, and SIK2 has recently been discovered to participate in the regulation of adipocyte autophagy via the transcription factor TFEB. Phosphorylation of TFEB by mTOR (mammalian target of rapamycin complex 1) results in its inactivity and subsequent downregulation of autophagy-related genes. We investigate here whether SIK2’s role in the regulation of TFEB is via mTOR signalling pathway, and do so by analyzing the effect of SIK inhibition on mTOR activity. We found that MRT199665, which is an AMPK and AMPK-related kinase inhibitor, results in inhibition of mTOR activity – seen as reduced phosphorylation of mTOR substrates ULK1 (Unc-51 autophagy activating kinase 1) and P70s6K (P70s6K-ribosomal protein s6 kinase). However, when SIKs were inhibited using SIK inhibitor HG-9-91-01, it resulted in reduced expression levels of these proteins, rather than altered phosphorylation. In human adipocytes, there was no significant mean difference between the negative control and treatment with HG-9-91-01. Overall, we can say that AMPK-related kinases, however not SIKs, appear to have a role to play in controlling mTOR activity.

English. The role of SIK2 in the regulation of the mTOR pathway in adipocytes Diabetes mellitus is a metabolic disorder, which results in elevated blood sugar levels due to insufficient secretion of insulin and reduced sensitivity of insulin in target tissues. There are different types of diabetes present with type 1 and type 2 diabetes being at two ends of a spectrum. Years of research have concluded that diabetes is often associated with conditions such as obesity, cardiovascular disorders and altered signalling of proteins, which often results in modified cellular processes. One of the important cellular processes which are modified is autophagy. Autophagy is a mechanism by which the cell degrades and recycles the damaged components of the cell. Autophagy is highly regulated, for example by the protein Transcription factor EB (TFEB), which is a protein required for autophagy. Adipose tissue is a major reservoir for storage of fat. Accumulation of fat in other tissues, which sometimes occurs during obesity, can give rise to complex diseases like diabetes. Insulin is an important signal for correct storage of fat in adipocytes, and insulin resistance in adipocytes could, therefore, contribute to incorrect fat storage in obesity. Dysfunctional autophagy is speculated to be one of several possible underlying triggers for insulin resistance in adipose tissue. SIK2 and autophagy Salt inducible kinases 2 (SIK2), an isoform of salt inducible kinases, is an AMPK-related kinase present in high quantities in adipose tissue. SIK2 is known to play a role in the regulation of metabolic processes but recent studies have also shown that SIK2 regulates TFEB activity. TFEB activity is regulated by the protein mammalian target of rapamycin (mTOR) which is also a master regulator of protein synthesis and cell growth in response to nutrient supply. mTOR phosphorylates TFEB and prevents its translocation to the nucleus which results in autophagy genes not being activated. Inhibition of mTOR allows activation of autophagy-related genes. With this as a background, we wish to see whether this regulation of TFEB by SIK2 is via mTOR or other pathways. Our results indicated that AMPK-related kinases do have a role to play in controlling mTOR activity but whether this is via SIK family is still doubtful since inhibition of SIKs did not result in reduced protein mTOR activity. Master’s Degree Project in Molecular Biology 45 credits 2020 Department of Biology, Lund University Advisor: Olga Göransson and Florentina Negoita Department of experiment medicine, Biomedical centre