The roles of S14 in endocrine and metabolic disorders:clinical and basic studies

• Main Authors: Yen-Ting Chen, 陳彥婷
• Other Authors: 楊偉勛
• Format: Others
• Language: en_US
• Published: 2019
• Online Access: http://ndltd.ncl.edu.tw/handle/979rh3

博士 === 國立臺灣大學 === 臨床醫學研究所 === 107 === Organs of endocrine system secret hormones, which circulate in blood, modulate physiological functions of end organs, and maintain body maintain homeostasis. Common endocrine disorders include thyroid dysfunction, diabetes mellitus, obesity, and etc. Thyroid which secrets thyroid hormones to affect rates of metabolism is a very important endocrine organ. S14 protein is mainly expressed in lipid-producing tissues, such as liver, mammary gland and adipose tissue. S14 is a thyroid hormone responsive gene which can be tremendously induced by T3, glucose, and insulin, and involves in fatty acid synthesis. However, the presence of S14 in human serum and its relation with thyroid function status have not been investigated. Compared to wild type, S14 knockout mice had resistance to diet-induced obesity and better glucose tolerance. The mRNA expression of S14 in the adipose tissue of obese subjects was lower than the controls. It was suppressible by fasting in non-obese subjects but not in the obese subjects. These findings suggested that S14 may participate in the pathogenesis of obesity and its co-morbidities in humans. However, the associations between serum S14 level and metabolic variables in humans have never been investigated. We developed an enzyme-linked immunosorbent assay (ELISA) for S14, and explored the possibility of using S14 as a biomarker in thyroid or metabolic disorders. In the first part of our clinical study, we compared the difference of serum S14 concentrations between patients with hyperthyroidism and euthyroidism. We further evaluated the associations between serum S14 and free thyroxine (fT4) or thyroid-stimulating hormone (TSH) levels. Twenty-six hyperthyroid patients and 29 euthyroid individuals were recruited. Data of all patients were pooled for the analysis of the associations between the levels of S14 and fT4, TSH, or quartile of TSH. Our analysis revealed that the hyperthyroid patients had significantly higher serum S14 levels than the euthyroid subjects. In univariate linear regression, the log-transformed S14 level (logS14) was positively associated with fT4 and negatively with TSH or quartile of TSH. The negative associations between logS14 and quartile of TSH remained significant in multiple linear regression. Our study is the first to investigate the serum S14 levels in humans and the effects of thyroid function statuses on serum S14 levels. The second part of our clinical study was designed to evaluate the associations between serum S14 concentrations with components of metabolic syndrome (MetS). A total of 327 subjects were recruited in this cross-sectional study and categorized by presence of MetS. Our analysis revealed that the patients with MetS had lower serum S14 levels than those without. Negative associations existed between MetS, central obesity, high triglyceride (TG), low high density lipoprotein C (HDL-C) and logS14. Our analysis also revealed a negative association of age with logS14. This is the first study to evaluate levels of S14 in patients with or without MetS. In basic study, the exact function of S14 remains largely unclear and the studies of S14 in the adipose tissues are rare. However, the expression level of S14 in adipose tissue is the most abundant among all tissues. We found that after induction of 3T3-L1 with the cocktail of differentiation medium, S14 mRNA was up-regulated by more than 700-fold. We also found that knockdown of S14 by transducing lentiviral S14 shRNA reduced 3T3-L1 adipocyte differentiation and lipid accumulation. In addition, we also found that many transcription factors involved in adipocyte differentiation pathway, such as Krüppel-like factor 15 (KLF15) and peroxisome proliferator-activated receptor (peroxisome proliferator-activated receptor gamma, PPARγ) were affected by the knockdown of S14 gene. According to the immunofluorescence staining data, we also observed that S14 protein is mainly expressed in the nucleus of 3T3-L1 pre-adipocytes, while mainly expressed in the cytoplasm in differentiated fat cells. Previous studies have shown that the S14 protein may be a transcription factor or co-activator. We tried to investigate whether S14 can directly or indirectly affect gene transcription. We performed luciferase reporter assay and found that the promoter activity of KLF15 and PPARγ with S14 overexpression constructs were slightly increased when compared to the control groups. We further used chromatin immunoprecipitation (ChIP) combined with high-throughput next-generation sequencing and general immunoprecipitation combined with mass spectrometry (IP-Mass) to search the potential S14 targeted-DNA sequences and potential S14 interacting proteins. In our preliminary study, we found that the interleukin (IL) 1β-treatment was capable of reducing adiponectin level, as well as S14 expression. In this study, we found that tunicamycin or thapsigargin induced endoplasmic reticulum stress (ER stress) may trigger the mRNA expressions of adiponectin and decrease S14. Previous studies have shown that mice lacking S14 gene have better insulin sensitivity. We intend to explore the regulation of S14 expression in inflammation or ER stress-related insulin resistance. In order to understand the role of S14 in adipose tissue, we have been working on tissue specific conditional S14 knockout mice under the service of Gene Knockout Mouse Core Laboratory of NTU enter of Genomic Medicine. We used CRISPR/Cas9 gene editing technology to insert loxp respectively at 5’ and 3’ to S14 exon 1. Then we bred this strain with adiponectin-CreERT mice which was purchased from the Taiwan’s National Laboratory Animal Center and selected to perform the adipose tissue-specific S14 knockout strain to investigate the function of S14 in adipose tissue. This part of the experiment is still in progress. Based on the above results, we successfully developed an enzyme immunoassay to measure the concentration of S14 in human serum, and applied it to explore the relationship between human S14 and thyroid function as well as metabolic syndrome. We found that the serum concentration of human S14 was negatively correlated with TSH. In addition, the concentration of S14 in the blood of patients with obesity or metabolic syndrome was significantly lower than that of normal subjects. These data are consistent with previous animal studies. We hope that in the future, this system can be used to explore more human diseases related to insulin resistance or fat synthesis, such as type 2 diabetes or non-alcoholic steatohepatitis. At the same time, we also confirmed by cell experiments that S14 participates in the adipocyte differentiation and oil droplet synthesis. We found that S14 may indeed involve in the inflammatory response- or endoplasmic reticulum stress- induced insulin resistance. However, the detail mechanisms require more investigations in the future.