In vitro Type Two Diabetes Mellitus Tissue Model to Investigate Insulin Resistanc

描述(由申请人提供)：本提案的目标是为2型糖尿病(T2 DM)开发一种与生理相关的3D组织系统，T2 DM是一种以胰岛素抵抗为特征的疾病状态。虽然脂肪组织不是人类葡萄糖处置的主要部位，但T2 DM患者的葡萄糖摄取、脂肪因子分泌和脂肪分解都发生了变化。胰岛素抵抗脂肪组织中脂肪分解的增加可以通过分泌游离脂肪酸(FFAs)直接导致肝脏和骨骼肌的胰岛素抵抗，FFAs激活了已知的破坏胰岛素信号的途径。动物模型对肥胖和2型糖尿病都有很好的认识，但人类和啮齿动物脂肪组织功能之间存在一些重要的差异，因此有必要从人类细胞衍生出相关的体外模型。3D组织被认为是体外疾病模型所必需的(Bin Kim 2004；Sainz 2009；Marrero 2009；Bott 2010)，但这些研究并没有集中在脂肪组织上。目前尚不清楚3D培养是否会比2D培养更好地代表体内脂肪组织，但我假设3D培养的内皮细胞将比2D培养得更好，3D培养是随后组织成管腔所必需的。3D组织还被假设具有增强的细胞-细胞外基质(ECM)相互作用，这些细胞-细胞外基质(ECM)可能沉积在细胞周围。目的1研究人脂肪细胞与内皮细胞的2D和3D共培养，以确定不同几何形状的共培养在结构和功能上的基线差异。AIM 2将通过整合人类成纤维细胞和单核细胞来增加模型的复杂性，这些细胞可以激活M1巨噬细胞，创造出像肥胖脂肪组织一样的促炎环境。这些培养物的特征包括：DNA定量，qRT-PCR相对基因表达，ELISA分泌蛋白质，比色法甘油三酯积累和脂肪分解，Western印迹相对蛋白质表达，葡萄糖和乳酸测定能量消耗。显微镜(光、荧光、共聚焦)将用于检查细胞组织、脂肪细胞大小和脂肪堆积。对激素的功能反应将评估生理反应。AIM 3将把在AIM 2中开发的2D和3D培养暴露于胰岛素和游离脂肪酸，这两种刺激在肥胖的2型糖尿病患者中升高，以及改变ECM重塑的蛋白质。组织对T2 DM刺激和改变的重塑条件的反应将首先单独比较，然后一起比较。已知受游离脂肪酸和炎性细胞因子影响的胰岛素信号通路的激活也将用Western印迹法检测，以确定通路激活如何随着这些刺激和几何形状的变化而变化。因此，这项拟议的工作将通过为发炎的脂肪组织开发新的3D体外模型，直接比较2D和3D培养的结构和功能，以及通过了解胰岛素信号如何在肥胖2型糖尿病患者中发现的刺激反应中受损，以及在2D和3D模型中可能改变以及改变的基质重塑，来促进对脂肪组织工程的理解。

DESCRIPTION (provided by applicant): The goal in the present proposal is to develop a physiologically-relevant 3D tissue system for type two diabetes mellitus (T2DM), a disease state hallmarked by insulin resistance. While adipose tissue is not the primary site for glucose disposal in humans, glucose uptake, secretion of adipokines, and lipolysis are all altered in T2DM. Increased lipolysis in insulin resistant adipose tissue can directly contribute to insulin resistance in liver and skeletal muscle through secretion of free fatty acids (FFAs) that activate pathways known to disrupt insulin signaling. Animal models have given great insight to both obesity and T2DM, but there are several important differences between human and rodent adipose tissue function that necessitate the development of a relevant in vitro model derived from human cells. 3D tissues are proposed to be essential for in vitro disease models (Bin Kim 2004; Sainz 2009; Marrero 2009; Bott 2010), but these studies have not focused on adipose tissue. It is not known if a 3D culture will better represent in vivo adipose tissue than 2D, but i is hypothesized that endothelial cells will polarize better in 3D than 2D, and that 3D is necessary for subsequent organization into lumens. The 3D tissue is also hypothesized have enhanced cell-extracellular matrix (ECM) interactions from a greater amount of ECM that may be deposited around the cells. Aim 1 will investigate 2D and 3D co-cultures of human adipocytes with endothelial cells to establish baseline differences in structure and function of the co- cultures in the different geometries. Aim 2 will add complexity to the model by incorporating human fibroblasts and monocytes, which may activate to M1 macrophages to create a pro-inflammatory environment like that found in obese adipose tissue. The cultures will be characterized by quantifying DNA, relative gene expression by qRT-PCR, secreted proteins by ELISAs, triglyceride accumulation and lipolysis using colorimetric assays, relative protein expression by Western blot, and energy use by glucose and lactate assays. Microscopy (light, fluorescence, confocal) will be used to examine cellular organization and adipocyte size and lipid accumulation. Functional responses to hormones will assess physiological responses. Aim 3 will expose the 2D and 3D cultures developed in Aim 2 to insulin and FFAs, two stimuli elevated in obese type 2 diabetics, and proteins that alter ECM remodeling. The tissue responses to the T2DM stimuli and the altered remodeling conditions will be compared separately first and then together. Activation of insulin signaling pathways known to be affected by FFAs and inflammatory cytokines will also be examined using Western blot to determine how pathway activation may change in response to these stimuli and with geometry. The proposed work will thus advance understanding of adipose tissue engineering by developing a new 3D in vitro model for inflamed adipose tissue, directly comparing structure and function of 2D and 3D cultures, and by understanding how insulin signaling is impaired in response to stimuli found in obese type 2 diabetics and may change in 2D and 3D models and with altered matrix remodeling.