Type Two Diabetes Mellitus Tissue Model to Investigate Insulin Resistance

描述（由申请人提供）：本提案的目标是为2型糖尿病（T2DM）开发一种生理相关的3D组织系统，T2DM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体葡萄糖处理的主要部位，但葡萄糖摄取、脂肪因子分泌和脂肪分解在T2DM中都发生了改变。胰岛素抵抗脂肪组织中增加的脂肪分解可以通过分泌游离脂肪酸（FFAs）直接促进肝脏和骨骼肌的胰岛素抵抗，游离脂肪酸（FFAs）激活已知的破坏胰岛素信号的途径。动物模型已经为肥胖和2型糖尿病提供了很好的见解，但是人类和啮齿动物脂肪组织功能之间存在一些重要差异，需要开发来自人类细胞的相关体外模型。三维组织被认为是体外疾病模型的必要条件（Bin Kim 2004; Sainz 2009; Marrero 2009; Bott 2010），但这些研究并未关注脂肪组织。目前尚不清楚3D培养是否比2D更好地代表体内脂肪组织，但假设内皮细胞在3D中比2D中极化更好，并且3D对于随后组织成管腔是必要的。3D组织也被假设增强了细胞外基质（ECM）的相互作用，因为大量的ECM可能沉积在细胞周围。目的1将研究人类脂肪细胞与内皮细胞的二维和三维共培养，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将通过纳入人成纤维细胞和单核细胞来增加模型的复杂性，这可能会激活M1巨噬细胞，从而创造一个像肥胖脂肪组织中发现的促炎环境。培养将通过定量DNA、qRT-PCR的相对基因表达、elisa的分泌蛋白、比色法的甘油三酯积累和脂肪分解、Western blot的相对蛋白表达以及葡萄糖和乳酸测定的能量使用来表征。显微镜（光，荧光，共聚焦）将用于检查细胞组织和脂肪细胞大小和脂质积累。对激素的功能性反应将评估生理反应。Aim 3将把Aim 2中培养的2D和3D培养物暴露于胰岛素和FFAs中，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T2DM刺激的反应和改变的重塑条件将首先单独比较，然后一起比较。已知受FFAs和炎症细胞因子影响的胰岛素信号通路的激活也将使用Western blot来检查，以确定通路激活如何响应这些刺激和几何变化。因此，通过开发一种新的炎症脂肪组织的体外3D模型，直接比较2D和3D培养的结构和功能，以及通过了解肥胖2型糖尿病患者对刺激的反应中胰岛素信号是如何受损的，并可能在2D和3D模型中发生变化，并改变基质重塑，所提出的工作将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. PUBLIC HEALTH RELEVANCE: Obesity is a known risk factor for type 2 diabetes mellitus (T2DM), the most common form of diabetes. As the prevalence of T2DM is increasing due to obesity and the underlying mechanisms of insulin resistance are not well known, the proposed work seeks to develop a 3D tissue model for obese adipose tissue in humans to understand how excess nutritional and inflammatory signals lead to insulin resistance and to provide a physiologically- relevant platform that may be used to evaluate new therapies. This 3D tissue model approach is therefore anticipated to provide improved and novel insight into disease mechanisms, and thus treatment options, particularly when compared to currently used cell culture systems or animal models.