摘要：背景—我們近來首次發現分佈於左心房心室間（左迴旋冠狀動脈及大冠狀靜脈所在處）的局部心包膜脂肪（epicardialadiposetissue,EAT）厚度是唯一與包括代謝症候群危險因子、脂肪激素（adipokines）、發炎指標、及冠狀動脈硬化程度相關的心包膜脂肪度量。此相關性在控制腹內脂肪量及體質量指標後仍存在。我們亦發現 EAT 分泌之發炎蛋白及脂肪激素（resistin, visfatin, Sfrp5）量為皮下脂肪的 5-10 倍。經由應用本團隊所獨有之具微細胞解析度之最低侵入性倍頻顯微技術（harmonic-generation microscopy）及新合成之人類胰島素-金奈米（insulin-Au nanodots [insulin-Au ND]）顆粒，我們首次觀察到，與非冠心病病患相較，接受冠脈繞道手術之冠心病患，其 EAT 中的脂肪細胞大小分布變異較大、有較多（~5-10%）的小型脂肪細胞（直徑<40 m m）、且其脂肪細胞較不會攝入 insulin-Au ND、特別在脂肪細胞內的脂肪顆粒（lipid droplets）胞器內的insulin-AuND 量尤低，這些均可能與胰島素阻抗現象有關。在此三年計畫中，我們不僅要進一步觀察上述之 EAT 胰島素攝入變化是否有位置特異性及相關訊息傳遞分子和細胞代謝狀態變化（利用螢光代謝造影），我們還將利用吾人之獨特技術觀察循環於周邊血液中之白血球及其亞群的胞內胰島素阻抗現象（cellular insulin resistance）並探究其與動脈硬化疾病指標之相關性。本計畫的研究結果將使我們能更早期診斷出體內胰島素阻抗狀態，並進一步優化醫學界對相關動脈硬化疾病的診治。研究方法及預期結果—在此三年計畫之前一年半，我們計劃利用上述之嶄新技術，針對 80 位欲接受冠狀動脈繞道手術之冠心病病患及 40 位非冠心病病患欲接受瓣膜手術者（其中半數為糖尿病患），探究（1）不同部位之心包膜脂肪組織、縱膈脂肪組織、及皮下脂肪組織之脂肪細胞大小分佈、小型脂肪細胞（直徑<40 m m）比率、細胞代謝狀況、及細胞和其脂肪顆粒胞器之 insulin-AuND 攝入狀況及（2）循環血液中之白血球及其亞群血球的 insulin-Au ND 攝入狀況。倍頻顯微技術之觀察結果將以穿透式電子顯微鏡驗證。上述之血球及脂肪細胞度量將與脂肪組織或細胞培養液中之各種細胞激素數值（adiponectin, resistin, leptin, IL-6, IL-8, TNF- a , monocyte chemoattractant protein-1,macrophageinflammatoryprotein-1 b,granulocytecolonystimulatingfactor,IL-1ra,Sfrp5,PGC-1 a andUCP-1）、臨床代謝指標、血清脂肪激素/發炎指標、及冠狀動脈硬化指標進行相關性分析以探究其臨床意義。在本研究中，我們將會對每位病患於術前抽取靜脈血液及摘取四個不同部位的心包膜脂肪組織，包括三條冠狀動脈近端周圍的脂肪組織及右心室前表面之脂肪組織。 後一年半，我們將（1）分析不同部位脂肪細胞內之各種訊息傳遞分子表現量，並探究其與 insulin-Au ND 攝入度量及細胞代謝狀況之相關性，（2）分析循環白血球及其亞群之胞內訊息傳遞分子表現量及與 insulin-AuND 攝入度量之相關性，及（3）評估循環白血球及其亞群之 insulin-Au ND 攝入度量是否能作為評估心血管風險之診斷工具。組織及培養之脂肪細胞將利用分層技術根據細胞大小分成不同亞群獨立觀察分析。不同細胞之 insulin-AuND 攝入度量將與胰島素受體相關分子（insulin-receptor,insulinreceptorsubstrate-1, caveolin-1, Akt, PPAR g, and SREBP-1C）、脂肪顆粒相關分子（perilipin-1,CGI-58,CIDEC,cavin-1,hormone-sensitivelipase,andATGL）、及發炎相關分子等進行機轉探討。臨床意義—本計畫利用本團隊獨創之倍頻顯微技術結合 insulin-AuND 首次可直接觀察血球細胞及脂肪細胞內之胰島素代謝過程，配合新的細胞代謝螢光造影技術，可謂在胰島素阻抗/動脈硬化領域之突破性研究。此研究結果不僅可能開啟調控脂肪組織進而治療包括胰島素阻抗、糖尿病及動脈硬化疾病之契機，其於白血球細胞之檢測技術開發，更可能轉變目前對胰島素阻抗及動脈硬化疾病的診治觀念。
Abstract: Background—We have first demonstrated that thickness of epicardial adipose tissue (EAT) in the left atrioventricular groove is the only EAT measurement significantly associated with the metabolic syndrome, systemic inflammatory markers, and coronary atherosclerosis after adjustments for intra-abdominal adiposity. We also show that EAT is associated with enhanced release of various inflammatory markers and adipokines. By applying the unique harmonic-generation microscopy (HGM) optical virtual biopsy system with subcellular resolution and fluorescent human insulin-Au nanodots (ND), we further demonstrate that, compared to patients with no cardiovascular risk factors, patients with coronary artery disease (CAD) have wider variation in the size of epicardial adipocytes and decreased insulin uptake in adipocytes, particularly at the intracellular lipid droplets level. In this 3-year project, we will not only explore the topographic variation of the above-mentioned characteristics of epicardial adipocytes, their cellular metabolic status by an novel fluorescent metabolic imaging, and their downstream signaling pathways, but also extend to investigate the extent of cellular insulin resistance in circulating leukocytes and their subpopulations to further advance the recognition and management of insulin resistance and atherosclerotic diseases. Methods and Expected Results—In the first 1.5 years of this 3-year research project, we plan to extend our previous observations by (1) applying the HGM to analyze whether there is regional variation in the proportion of small-sized (<40 mm) adipocytes, the cellular insulin-Au ND uptake profile, and metabolic status in EAT; and (2) investigating the the cellular insulin-Au ND uptake profile of circulating leukocytes and their subpopulations obtained from CAD patients undergoing bypass surgery and non-CAD patients undergoing valve surgery, either with or without diabetes in a 2 x 2 design. 80 CAD patients and 40 age, gender, and body mass index-matched non-CAD patients will be included. Correlation analyses of the cellular (adipocyte or leukocyte) measurements from the above analyses with cytokine/adipokine profiles (adiponectin, resistin, leptin, IL-6, IL-8, TNF-a, monocyte chemoattractant protein-1, macrophage inflammatory protein-1b, granulocyte colony stimulating factor, IL-1ra, Sfrp5, PGC-1a and UCP-1) from adipose tissue-conditioned media, various systemic metabolic parameters, serum markers of inflammation and adipokines, and parameters of global and individual coronary atherosclerosis will be done to elucidate their clinical significance. In the second 1.5 years of this 3-year research project, we will (1) assess the expressions of various signaling molecules in adipocytes of different size among patients with different disease status and whether these molecules are related to the trafficking of insulin-Au in and out of adipocytes and intracellular lipid droplets, not only in human surgical specimens, but also in cultured 3T3-L1 adipocytes; (2) investigate the correlation of expression profile of intracellular signaling pathway molecules with the insulin uptake status in circulating leukocytes and their subpopulations to elucidate their pathophysiological significance; and (3) assess the clinical utility of leukocyte functional analysis for risk-stratification and therapeutic guidance. We will perform molecular analyses (western blotting and quantitative reverse transcriptase-polymerase chain reaction [RT-PCR], etc.) to assess the expressions of insulin-receptor-related molecules (insulin-receptor, insulin receptor substrate-1, caveolin-1, Akt, PPARg, and SREBP-1C), lipid-droplet-related molecules (perilipin-1, CGI-58, CIDEC, cavin-1, hormone-sensitive lipase, and ATGL), and inflammation-related molecules in human adipocytes of different sizes by cell fractionation, 3T3-L1 adipocytes of different sizes, and different leukocyte subpopulations. Clinical Significance—This 3-year research project will be a breakthrough in the field of insulin resistance and atherosclerosis research as we are for the first time able to directly measure insulin sensitivity of circulating leukocyte and its subpopulations. Results from this project will provide us opportunities to ameliorate/prevent diseases from insulin resistance to atherosclerosis by early identification of cellular insulin resistance and modulating the “crazy” visceral adipose tissues at the molecular level. Methodological breakthrough established in this project can be translated to other fields of medical research and even be applied to clinical practice (for example, leukocyte insulin sensitivity functional analysis as a better marker of insulin sensitivity).