SIK2 mutagenic 3T3-L1을 이용하여 나트륨이 adipogenesis에 미치는 영향에 관한 연구

Abstract

Obesity, an imbalance between energy intake and expenditure, is a risk factor of various metabolic diseases. Being obese starts with the increase of number of body fat and leads to the adipocyte hypertrophy. Malfunction of adipose tissue results in increased insulin resistance and metabolic functions. Numerous studies have shown that high-salt intake is associated with obesity, insulin resistance, type 2 diabetes, and cardiovascular disease. However, further studies to figure out metabolic pathway related to salt is needed. Salt-inducible kinases(SIKs), serine/threonine kinase, are one of the AMP-activated protein kinase(AMPK) family. Recently, its diverse functions were known to regulate salt homeostasis and metabolism among many tissues. SIK2 is abundantly expressed in adipocytes and its role is crucial. Here, it is to find what SIK2 mutagenic 3T3-L1 cell affects on adipogenesis when salt is treated. 3T3-L1 cells were cultured throughout this study. MTT assay was done to figure out cell viability on salt in pre-adipocyte. Furthermore, oil Red O staining was administrated in adipocyte. Salt concentration was determined to be 50mM through two experiments. Ubiquitin is a small protein found in almost all tissues of eukaryotes, acting as a post-translational modification. Ubiquitylation is the process of attaching ubiquitin to other target proteins. As ubiquitin has its own lysine, it binds well to lysine residues in the gene sequence and causes degradation or acts as a regulator of protein activity. In order to exclude the effect of ubiquitin, K should be changed to Q in gene sequence. In this study, K positions of 536 and 702 of the SIK2 gene sequence were transformed to Q. SIK2 mutagenic 3T3-L1 cells were transfected by electroporation method using Lonza. Four cells were set up; 3T3-L1(control), SIK2 K536Q, SIK2 K702Q, SIK2 KO(negative control). Western blotting analysis was used to figure out the expression level of SIK2 and adipogenesis related factors, PPARγ and C/EBPα. The expression level of SIK2 in K702Q was not changed when compared to the control. K536Q was similar to KO and the activity was decreased compared to K702Q. Adipogenesis factors were decreased in expression compared to the control. In comparison with SIK2 mutagenic 3T3-L1, K536Q exhibits low expression of SIK2 but high in adipogenesis factors, however, K702Q expressed opposite results. mRNA microarray analysis were done on four cells. By using gene ontology of cell differentiation, each genes were categorized. The categorized genes were then selected on the standard of fold change comparing to control. Genes were analyzed by using KEGG mapper to figure out each metabolic pathways. Real-time PCR were used to compare expression level on each cells when salt is treated or not treated. Particularly in K702Q cell, cAMP signaling pathway related genes, NR4A2, MED1, increased when salt is treated. Moreover, expression of ER stress related genes, Bscl2 and Eif2ak3, increased when high in salt. However, negative regulator of TGF-β signaling pathway, Smad6 and CTBP1, decreased when salt is treated. This study showed that K702Q, SIK2 activated cell, is related to adipogenesis when salt is treated. However, K536Q, SIK2 less activated cell, does not have correlation with adipogenesis when salt is treated. As the standard of SIK2 mutagenic cells was ubiquitylation, there were various genes related to different metabolic pathways. As obesity occurs from various environmental factors, race, and sex, it is necessary to confirm diverse obesity phenotype. In particular, it is expected that this study will be used as a basis for the study of the mechanism of obesity induced by salt intake.