S14在內分泌及代謝疾病中的角色:臨床與基礎研究

Abstract

內分泌系統為無導管腺體，藉由分泌激素去調控動物體內各種生理功能之正常運作，是維持體內恆定的重要控制系統。臨床上常見的內分泌疾病包括糖尿病、甲狀腺疾病以及肥胖症等，其中甲狀腺所分泌的甲狀腺素會去調控身體每個器官的新陳代謝，是極為重要的內分泌腺體。 S14蛋白主要在會製造脂肪的組織表現，例如肝臟、乳腺和脂肪組織，參與脂肪酸合成，在大鼠肝臟中會被三碘甲狀腺激素(T3)大量誘發。然而，尚未有研究報告人血清中S14的存在及其甲狀腺功能狀態的關係。另外，動物實驗顯示S14基因發生缺失(deletion)時，小鼠對飲食誘導的肥胖和葡萄糖耐受具有更好的抗性，暗示S14基因在脂肪代謝中，扮演一定的角色。最近的研究顯示S14的傳訊核醣核酸(mRNA)在肥胖的個體中，其表達量減少，且禁食無法抑制其表達量，暗示S14可能參與肥胖及其共患疾病的病生理機轉，但目前尚未有研究探討人類血清中的S14與代謝相關因子之間的關聯性。我們利用自行開發的酵素免疫分析法偵測S14 在血清中的濃度變化，探討S14作為生物標誌物應用於甲狀腺疾病、肥胖或是內分泌代謝相關疾病的可能性。 我們在臨床研究的第一部分，比較甲狀腺功能亢進症與甲狀腺功能正常患者的血清S14濃度並且評估血清S14與游離型甲狀腺素（fT4）或促甲狀腺激素（TSH）之間的關聯。我們招募了26名甲狀腺功能亢進患者和29名甲狀腺功能正常的人，分析S14和fT4、TSH或TSH四分位數之間的關聯。我們的分析顯示，甲狀腺功能亢進患者的血清S14濃度顯著高於甲狀腺功能正常的患者。在單變量線性回歸中，對數轉換的S14濃度（logS14）與fT4 呈現正相關且與TSH或TSH四分位數呈現負相關。在多變量線性迴歸分析中，logS14和TSH四分位數之間的負相關仍然顯著。我們的研究首先證實了甲狀腺功能狀態對人類血清S14濃度的影響。 我們臨床研究的第二部分招募了總共327名受試者，依據其是否有代謝症候群進行分類。我們的分析顯示，患有代謝症候群患者的血清S14濃度低於沒有代謝症候群的患者。代謝症候群、中心性肥胖、高三酸甘油酯、高密度脂蛋白低下和logS14之間存在負相關。我們的分析還揭示了年齡與logS14的負相關。這是第一篇探討人類代謝症候群與血清S14濃度是否相關的研究。 在基礎研究方面，S14蛋白的作用機轉目前還不明確，且S14在脂肪組織的研究仍然很少。然而，S14在脂肪組織之表現量其實是在各種組織中表現量最高的。我們發現隨著3T3-L1的分化，S14基因的mRNA表現量也會跟著上升。如果在3T3-L1前脂肪細胞(pre-adipocyte)中削弱S14基因的表現，會抑制脂肪細胞分化並影響其油滴的聚集。此外我們也發現許多參與在脂肪細胞分化途徑中重要的轉錄因子，例如 Krüppel-like factor 15(KLF15)以及過氧化物酶體增殖物活化受體(peroxisome proliferator-activated receptor gamma, PPARγ)也會因削弱S14基因表現而受到影響。我們也藉由免疫螢光染色的方式觀察到S14蛋白主要表現在3T3-L1前脂肪細胞的細胞核內，而在分化完成的脂肪細胞主要卻是表現在細胞質中。由於先前有研究顯示S14蛋白可能是一個轉錄因子或是轉錄共同激活因子，為了瞭解S14是否可以直接或是間接影響基因的轉錄，我們利用螢光素酶報告基因測定法(luciferase reporter assay)發現，相較於對照組，雖然KLF15以及PPARγ啟動子被激活的倍數不高，但在有S14表達載體的伴隨下還是有上升的趨勢。同時我們利用染色質免疫沉澱法(chromatin-immunoprecipitation, ChIP)合併高通量次世代定序(high-throughput next generation sequencing)以及一般的免疫沉澱法合併質譜儀分析(IP-Mass)去找尋S14蛋白可能作用的DNA序列以及直接或是間接有相互作用的蛋白質。此外，在本實驗室的前期研究當中發現當脂肪細胞處以促發炎的白細胞介素1β型(interleukin 1β, IL-1β)時，除了脂締素(adiponectin)這個與胰島素阻抗極為相關的因子的基因表現量會下降外，S14的基因表現也是極為顯著的下降。同時我們在這個研究當中也發現由衣黴素(tunicamycin)或是毒胡蘿蔔素(thapsigargin)所誘導的內質網壓力(endoplasmic reticulum stress, ER stress)同樣也會造成脂締素及S14 mRNA表現量下降。由於先前研究指出S14基因缺失的老鼠會有較好的胰島素敏感性，因此S14在發炎或是內質網壓力所引起的胰島素阻抗之調控也是未來我們想探討的議題之一。 為了進一步了解S14在脂肪組織的角色，我們與台灣大學基因體研究中心轉殖動物核心實驗室合作，利用CRISPR/Cas9 基因編輯技術將能轉錄出S14 蛋白質的外顯子前後插入loxp，並與由國家動物中心購入的脂締素-CreERT 老鼠配種，目的是選育出能針對脂肪組織施行特定時間剃除S14 基因的小鼠來釐清S14在脂肪組織的角色，這部分的實驗依然在進行當中。 綜合以上結果，本研究成功地開發可以測定人體血清中S14濃度的酵素免疫分析法，並且真實地應用在探討人體S14與甲狀腺功能以及代謝症候群之間的相關性。我們發現人體S14血清濃度與促甲狀腺激素成顯著負相關，此外，肥胖或是患有代謝症候群的患者其血中S14濃度顯著低於正常的組別，這些結果皆與前人的動物研究相呼應。我們希望未來這一套酵素免疫分析法能用來探討人類更多與胰島素阻抗或是脂肪生合成相關的疾病，例如第二型糖尿病或是非酒精性肝炎。同時，我們也藉由細胞實驗證實S14會參與在脂肪細胞分化以及油滴生合成的機轉當中，並且發現S14的確可能參與在發炎反應或是內質網壓力所誘發的胰島素阻抗，然而更詳細的機制還需要後續更多的研究來證實與闡釋。

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.