新型脂肪因子ITIH5调控脂肪功能障碍的作用及机制研究

Inter-α-trypsin inhibitor heavy chain 5 （ITIH5）is a recently discovered ITIH family member and studies have suggested its role in ECM stabilization due to its binding site to hyaluronan (HA). CD44 is one of the major receptors for HA and both HA and CD44 have been reported to play important roles in obesity-induced adipose tissue inflammation and insulin resistance. Our previous study has found a significant higher level of ITIH5 in adipose tissue of adult C57BL/6 mice and human primary adipocytes. Moreover, we found a higher ITIH5 mRNA levels in subcutaneous adipose tissue of HFD-induced obese mice, suggesting that ITIH5 could have a link to obesity. In the current study, we propose to study the role and molecular mechanism of ITIH5 in adipose cells and obesity-induced adipose tissue dysfunction. We will firstly study the mRNA and protein expressions of ITIH5 in three parts of adipose tissue (epididymal, subcutaneous and brown) of WT C57BL/6 mice and HFD-induced obese mice and determine the ITIH5 expressing cell types in adipose tissue by immunohistochemistry staining. After that, a correlation analysis will be conducted to examine the relationship between adipose ITIH5 expressions and obesity-associated metabolic parameters in serum such as fasting glucose, fasting insulin, adiponectin/leptin levels and inflammatory cytokine levels. Secondly, We will test the effect of ITIH5 overexpression on preadipocytes proliferation, differentiation and TGF-β1-induced fibrotic responses. Also, we will test whether knockdown or overexpression of ITIH5 in adipocytes affects their insulin sensitivity and endocrine function under normal (no treatment) and inflammatory conditions (co-culture with macrophages or treated with inflammatory cytokine IL-1 β). Thirdly, since HA/CD44 is a potential binding partner to ITIH5, we will test whether HA/CD44 was mediating the effects of ITIH5 on preadipocyte/adipocyte functions. We will examine the colocalization of ITIH5 to HA and CD44 by Co-IP and immunoflurescence staining in both ITIH5 overexpressed preadipocytes and adipocytes in normal and inflammatory conditions. Then, we will test whether downregulation of HA and CD44 could counteract the effect of ITIH5 overexpression on preadipocytes and adipocytes functions in normal and inflammatory conditions. Finally, we will use the adipose specific ITIH5 knockout mice and fed the mice with HFD for up to 16 weeks and examine the parameters associated with obesity (e.g. body weight, adipose tissue weight, fating glucose and insulin etc), adipose tissue inflammation (macrophage infiltration, type and numbers), insulin resistance, adipose tissue HA/CD44 expressions, and adipose tissue fibrosis (PDGFRα+CD9high cell population, α-SMA expression and TGFβ signaling activation etc). In summary, the proposed study will provide the scientific evidence for identifying the ITIH5 as a new adipokine and elucidating its potential role and mechanism in regulating adipose tissue function in obesity.

间胰蛋白酶H5重链（ITIH5）可结合透明质酸（HA）并与CD44结合参与肥胖引起的脂肪组织炎症和胰岛素抵抗。我们发现ITIH5在人脂肪细胞和高脂诱导的肥胖小鼠皮下脂肪中显著升高，但其作用不明。故本项目拟先在高脂诱导的肥胖小鼠多种脂肪组织中确定ITIH5的表达水平、细胞定位及与肥胖相关代谢指标的相关性。再用炎性因子或巨噬细胞处理ITIH5高/低表达的原代人前/脂肪细胞，检测前脂肪细胞增殖、分化与纤维化、脂肪细胞胰岛素敏感性和脂肪因子分泌；用Co-IP和免疫荧光明确ITIH5与HA/CD44是否共定位；通过降低HA/CD44明确ITIH5调控前/脂肪细胞功能的分子机制。另对脂肪特异性ITIH5敲除小鼠饲喂高脂诱导肥胖以验证其在肥胖中的作用及机制。本研究为阐明ITIH5调控脂肪细胞功能参与肥胖导致的脂肪功能障碍的作用及机制，并为将其作为一个潜在的肥胖及相关代谢疾病防治的靶点提供理论和实验依据。

ITIH5与肥胖相关指标BMI、血浆总甘油三酯和胰岛素水平及HOMA-IR相关，但其在肥胖中的作用不清。本课题利用小鼠肥胖模型和原代人的脂肪细胞探讨ITIH5与肥胖的关系及在脂肪细胞中的作用和分子机制。我们首先用LC-MS/MS对野生型小鼠和db/db糖尿病肥胖小鼠附睾周围脂肪组织分泌蛋白进行蛋白质组学分析，发现ITIH5的水平在db/db小鼠脂肪组织中显著降低。对成年雄性C57BL/6小鼠各种组织（如脂肪、肝、肺、肾、骨骼肌、胰腺、脾、动脉、脑和胃等）进行分析，发现ITIH5 mRNA水平在白色和棕色脂肪中具显著高表达。利用高脂诱导的肥胖小鼠模型，我们发现肥胖小鼠皮下脂肪中ITIH5 mRNA水平升高。进一步，我们发现ITIH5 mRNA水平在高脂肪饲喂的小鼠脂肪细胞和肥胖儿童脂肪组织中也显著升高。另外，ITIH5 mRNA和蛋白水平在第9天分化成熟的人白色脂肪细胞中显著升高，提示ITIH5是一种潜在脂肪因子，可能与白色脂肪细胞分化有关。此外，我们发现与ITIH家族其它分子相比，ITIH5是脂肪细胞唯一高表达的分子。最后，我们构建了全身性和脂肪特异性ITIH5 基因敲除小鼠。ITI5 KO小鼠在正常饲料饲喂时体重与对照组无显著差异。另外，我们发现致炎型巨噬细胞可下调脂肪细胞ITIH5的表达，提示其可能作为一个抗炎因子参与肥胖引起的慢性炎症。以上数据提示ITIH5是潜在的脂肪分泌蛋白，其表达水平在肥胖脂肪组织中升高可能作为肥胖的生物标志物（biomarker）。