Type Two Diabetes Mellitus Tissue Model to Investigate Insulin Resistance

用于研究胰岛素抵抗的二型糖尿病组织模型

• 批准号: 8315083
• 负责人: Kelly Anne Burke
• 金额: $ 4.92万
• 依托单位: TUFTS UNIVERSITY MEDFORD
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-04-01 至 2015-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8315083
• 关键词: Adipocytes; Adipose tissue; Affect; Animal Model; Biological Assay; Cell Culture System; Cell Polarity; Cells; Coculture Techniques; Collagen Type IV; Complex; DNA; Deposition; Development; Diabetes Mellitus; Diet; Disease; Disease model; Endothelial Cells; Environment; Exposure to; Extracellular Matrix; Fibroblasts; Fluorescence Microscopy; Functional disorder; Gelatinase B; Gene Expression; Glucose; Goals; Hormones; Human; Immune; In Vitro; Inflammatory; Insulin; Insulin Resistance; Insulin Signaling Pathway; Lead; Life Style; Lipids; Lipolysis; Liver; Matrix Metalloproteinases; Metabolic; Microscopy; Modeling; Non-Insulin-Dependent Diabetes Mellitus; Nonesterified Fatty Acids; Nutritional; Obesity; Overnutrition; Pancreas; Pathway interactions; Patients; Pattern; Physiological; Prevalence; Proteins; Relative (related person); Research; Risk Factors; Rodent; Signal Pathway; Signal Transduction; Site; Skeletal Muscle; Stimulus; Stromal Cells; Structure; System; TNF gene; Tissue Engineering; Tissue Inhibitor of Metalloproteinase-1; Tissue Model; Tissues; Triglycerides; Western Blotting; Work; adipokines; base; cell type; cytokine; diabetic; diabetic patient; experience; glucose disposal; glucose uptake; improved; in vitro Model; in vivo; insight; insulin sensitivity; insulin signaling; macrophage; monocyte; novel; obesity risk; protein expression; response; sedentary; three-dimensional modeling; tissue culture; tool

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.

描述（由申请人提供）：本提案的目标是开发一种用于治疗二型糖尿病（T2DM）的生理相关 3D 组织系统，二型糖尿病是一种以胰岛素抵抗为标志的疾病状态。虽然脂肪组织不是人体葡萄糖处理的主要场所，但 T2DM 中葡萄糖的摄取、脂肪因子的分泌和脂肪分解都发生了改变。胰岛素抵抗脂肪组织中脂解作用的增加可以通过分泌游离脂肪酸（FFA）来直接导致肝脏和骨骼肌中的胰岛素抵抗，从而激活已知破坏胰岛素信号传导的途径。动物模型对肥胖和 T2DM 提供了深入的了解，但人类和啮齿动物的脂肪组织功能之间存在一些重要差异，因此需要开发源自人类细胞的相关体外模型。 3D 组织被认为对于体外疾病模型至关重要（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 培养是否会比 2D 更好地代表体内脂肪组织，但我假设内皮细胞在 3D 中极化比 2D 更好，并且 3D 对于随后组织成管腔是必要的。还假设 3D 组织通过沉积在细胞周围的大量 ECM 增强了细胞与细胞外基质 (ECM) 的相互作用。目标 1 将研究人类脂肪细胞与内皮细胞的 2D 和 3D 共培养物，以确定不同几何形状的共培养物的结构和功能的基线差异。目标 2 将通过纳入人类成纤维细胞和单核细胞来增加模型的复杂性，这些细胞可能会激活 M1 巨噬细胞，以创建类似于肥胖脂肪组织中的促炎环境。通过定量 DNA、qRT-PCR 的相对基因表达、ELISA 的分泌蛋白、比色测定的甘油三酯积累和脂肪分解、蛋白质印迹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培养物。显微镜（光、荧光、共聚焦）将用于检查细胞组织、脂肪细胞大小和脂质积累。对激素的功能反应将评估生理反应。目标 3 会将目标 2 中开发的 2D 和 3D 培养物暴露于胰岛素和 FFA（肥胖 2 型糖尿病患者中升高的两种刺激物）以及改变 ECM 重塑的蛋白质。组织对 T2DM 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 FFA 和炎症细胞因子影响的胰岛素信号通路的激活也将使用蛋白质印迹进行检查，以确定通路激活如何响应这些刺激和几何形状而变化。因此，拟议的工作将通过开发一种新的发炎脂肪组织 3D 体外模型，直接比较 2D 和 3D 培养物的结构和功能，并了解胰岛素信号传导如何因肥胖中发现的刺激而受损，从而促进对脂肪组织工程的理解。 2 型糖尿病患者可能会在 2D 和 3D 模型中发生变化，并且会随着基质重塑的改变而发生变化。 公共卫生相关性：肥胖是 2 型糖尿病 (T2DM)（最常见的糖尿病形式）的已知危险因素。由于肥胖导致 T2DM 的患病率不断增加，并且胰岛素抵抗的潜在机制尚不清楚，因此拟议的工作旨在开发人类肥胖脂肪组织的 3D 组织模型，以了解过量的营养和炎症信号如何导致胰岛素抵抗并提供可用于评估新疗法的生理相关平台。因此，这种 3D 组织模型方法预计将提供对疾病机制的改进和新颖的见解，从而提供治疗选择，特别是与目前使用的细胞培养系统或动物模型相比。