亮氨酸通过脂肪组织/脂肪细胞控制体重的机制研究

Leucine, one branched-chain amino acid (BCAA), possesses many important biological functions. Our prevoius animal study found that leucine effectively promotes insulin resistance in rats. Furthermore, our INTERMAP study using diverse population samples in Japan, China, the UK and the US demonstrated that dietary leucine intake was significantly and inversely related to body mass index (BMI), which reflected the degree of obesity. Although it is well estabilished that leucine regulates ingestive behavior through the central nervous system (CNS), the effects of leucine on fat mass and adipocytes are still poorly understood. The purpose of current project is to determine the adipocyte differentiation, lipolysis and its metabolism including protein kinase A activity and following expression of perilipin and hormone-sensitive lipase, by the addition of different concentrations of leucine in 3T3-L1 cells, which differentiate into an adipocyte-like phenotype under appropriate conditions. Since leptin is secreted by adipocytes, the effects of leucine on leptin secretion and the regulation of leptin receptor activated JAK/ATATA signaling pathway are studied in 3T3-L1 cells. Then, the animals are injected with L-[1-13C]leucine solution into caudal vein and the distribution of leucine in adipocytes and other tissues will be observed using isotopic tracer technique. The long-term animal experiments will be performed using different dietary regimens: 1) normal rats are fed with leucine and high-fat diet; 2) obesity rats induced by high-fat diet are fed with leucine. On the other hand, the groups of partial deprivation of leucine are set as negative controls in both dietary regimens. The body weight of animal and energy intake are recorded weekly throughout the experiment. After decapitation, the distribution of white adipose tissue (WAT) and brown adipose tissue (BAT), and the amount and morphology of adipocytes will be examined. In addition, adipocyte metabolism and plasma biochemical parameters including amino acid profile, BCAA metabolic enzymes will be determined. Furthermore, leptin and JAK/ATATA signaling pathway will be analyzed with PCR and Western blot assays using primary cultured adipocytes. The innovation of this research is to investigate the effect of leucine intake on the control of body weight through adipose tissues and adipocytes, which is to provide mechanistic clue for the control of obesity and lifestyle-related diseases such as diabetes and cardiovascular diseases.

亮氨酸的营养新功能备受关注。申请人的前期工作发现亮氨酸可以改善模型大鼠的胰岛素抵抗，同时发现亮氨酸摄取与人群体质指数呈显著负相关。虽然已知亮氨酸通过中枢系统调节摄食行为，但尚不清楚其如何对体现肥胖程度的脂肪组织、脂肪细胞直接产生作用。本项目通过体外实验观察亮氨酸对3T3-L1脂肪细胞分解脂肪的作用及其分子机制，重点研究脂肪细胞分泌的瘦素及瘦素受体激活的JAK/STAT信号通路。在动物实验中，用核素标记观察亮氨酸在脂肪组织中的分布，然后采用1）高脂饲料和添加亮氨酸饲料同时喂养大鼠和2）高脂饲料诱导肥胖大鼠模型后再喂以添加亮氨酸的饲料，两组均设部分去除饲料中亮氨酸的对照组。从预防和治疗两个角度探讨亮氨酸对大鼠体重、白色和褐色脂肪组织的分布和形态、脂肪细胞代谢及原代培养脂肪细胞瘦素信号通路的影响，旨在为肥胖和糖尿病等慢性病的防治提供新思路。

研究发现亮氨酸通过中枢系统调节摄食行为，但尚不清楚其如何脂肪组织和脂肪细胞的直接作用。我们在高脂高胆固醇饲料的基础上添加1.5%和3.0%的亮氨酸干预C57BL/6J小鼠24周，发现亮氨酸干预显著降低了小鼠体重以及白色脂肪组织（WAT）中脂肪细胞的体积和肝脏中脂肪的堆积。亮氨酸干预下调了肝脏脂肪合成相关酶（FAS、ACC、SREBP-1、LXR-α）的蛋白表达，上调了WAT中脂肪分解相关酶（p-HSL、ATGL、）的表达并上调了WAT和褐色脂肪细胞中产热反应相关以及WAT褐色化相关蛋白（UCP-1、β-AR、PGC-1和FGF-21）的表达。因此，亮氨酸干预抑制了肝脏脂肪合成、促进脂肪组织的脂肪分解和WAT褐色化。接着，我们又探讨了亮氨酸对瘦素信号通路的影响。在高脂饲料的基础上添加1.5%、3%和4.5%亮氨酸连续干预SD大鼠16周。亮氨酸改善了脂肪变性，亮氨酸干预显著降低了血清瘦素水，但上调了下丘脑和脂肪组织中瘦素受体（ObR）的表达。另外，亮氨酸还上调了ObR激活的JAK2和STAT3蛋白表达，下调抑制瘦素信号通路的SOCS3的表达。因此，亮氨酸干预通过调节瘦素信号通路提高了瘦素敏感性。以上两个研究证实了高脂饲料喂养的同时添加亮氨酸能够通过脂肪细胞/脂肪组织起到抑制体重的作用。在人群干预中超重/肥胖的2型糖尿病患者每天补充3g亮氨酸3个月。与对照组比较，干预组体重、体脂、蛋白质和骨骼肌无显著差异，空腹血糖、糖化血红蛋白以及血脂与对照组也无显著差异。接着我们用亮氨酸直接干预手术过程中取出脂肪组织，发现甘油和游离脂肪酸水平和对照组无显著差异。亮氨酸也不影响HSL和ERK1/2信号通路。最后，我们用亮氨酸分别处理诱导分化过程中以及诱导分化完成后的3T3-L1前脂肪细胞。结果发现细胞分化过程中亮氨酸处理组的脂滴相对数低于对照组，分化完成后亮氨酸处理的各组脂滴相对数的差异无统计学意义。分化过程中采用亮氨酸处理，上调了p-HSL蛋白表达、下调了PLIN、瘦素和瘦素通路相关的ObR、JAK2和STAT3蛋白的表达。分化完成后亮氨酸处理，这些蛋白的表达量与对照组的差异无统计学意义。以上的动物实验、人群干预和细胞研究证实：亮氨酸摄取能预防肥胖发生，但对已形成的肥胖则无显著效果。