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

用于研究胰岛素抵抗的体外二型糖尿病组织模型

• 批准号: 8449320
• 负责人: Kelly Anne Burke
• 金额: $ 5.22万
• 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/8449320
• 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; 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; nutrition; 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.

描述（由申请人提供）：本提案的目标是开发一种用于2型糖尿病（T2 DM）（一种以胰岛素抵抗为特征的疾病状态）的生理相关3D组织系统。虽然脂肪组织不是人体中葡萄糖处置的主要部位，但T2 DM患者的葡萄糖摄取、脂肪因子分泌和脂解均发生改变。胰岛素抵抗性脂肪组织中增加的脂解可通过游离脂肪酸（FFA）的分泌直接导致肝脏和骨骼肌中的胰岛素抵抗，所述游离脂肪酸（FFA）激活已知破坏胰岛素信号传导的途径。动物模型对肥胖和T2 DM都有很好的了解，但人类和啮齿动物脂肪组织功能之间存在几个重要差异，需要开发源自人类细胞的相关体外模型。3D组织被认为是体外疾病模型所必需的（Bin Kim 2004; Sainz 2009;马雷罗2009; Bott 2010），但这些研究并未集中于脂肪组织。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中更好地生长，并且3D对于随后组织成管腔是必要的。还假设3D组织具有增强的细胞-细胞外基质（ECM）相互作用，来自可以沉积在细胞周围的更大量的ECM。目的1将研究人脂肪细胞与内皮细胞的2D和3D共培养物，以建立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，以创造一种类似于肥胖脂肪组织中发现的促炎环境。将通过定量DNA、qRT-PCR测定的相对基因表达、ELISA测定的分泌蛋白、比色测定的甘油三酯蓄积和脂解、Western印迹测定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。将使用显微镜（光学、荧光、共聚焦）检查细胞组织、脂肪细胞大小和脂质积聚。对激素的功能反应将评估生理反应。目标3将使目标2中开发的2D和3D培养物暴露于胰岛素和FFA，这两种刺激在肥胖2型糖尿病患者中升高，以及改变ECM重塑的蛋白质。首先分别比较对T2 DM刺激的组织反应和改变的重塑条件，然后一起比较。还将使用蛋白质印迹法检查已知受FFA和炎性细胞因子影响的胰岛素信号传导途径的活化，以确定途径活化如何响应于这些刺激和几何形状而改变。因此，拟议的工作将通过开发一种新的炎症脂肪组织的3D体外模型，直接比较2D和3D培养物的结构和功能，以及通过了解胰岛素信号传导如何响应肥胖2型糖尿病患者中发现的刺激而受损，并可能在2D和3D模型中发生变化以及改变基质重塑来促进对脂肪组织工程的理解。