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）：本提案中的目标是开发与二型糖尿病（T2DM）的生理相关的3D组织系统，这是一种疾病状态状态，该疾病状态是受胰岛素抵抗的标志性。虽然脂肪组织不是人类葡萄糖处置的主要部位，但T2DM中的葡萄糖摄取，脂肪分泌，脂肪因子的分泌和脂解会改变。胰岛素耐药脂肪组织中脂解的增加可以通过分泌自由脂肪酸（FFA）直接导致肝脏和骨骼肌的胰岛素耐药性，从而激活已知会破坏胰岛素信号传导的途径。动物模型对肥胖症和T2DM有了很大的了解，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，这些差异需要开发从人类细胞中产生相关的体外模型。有人认为3D组织对于体外疾病模型至关重要（Bin Kim 2004； Sainz 2009； Marrero 2009； Bott 2010），但这些研究并未集中在脂肪组织上。尚不清楚3D培养物是否比2D更好地代表体内脂肪组织，但我假设内皮细胞在3D中的偏振比2D优于偏振，并且3D对于随后的组织为流明是必不可少的。还假设3D组织从可能沉积在细胞周围的大量ECM中增强了细胞 - 细胞基质（ECM）相互作用。 AIM 1将研究与内皮细胞的人脂肪细胞的2D和3D共同培养物，以在不同几何形状中建立合作培养的结构和功能的基线差异。 AIM 2将通过掺入人体成纤维细胞和单核细胞来增加模型的复杂性，这些成纤维细胞和单核细胞可能会激活M1巨噬细胞以创建肥胖脂肪组织中的促炎环境。这些培养物的特征是通过使用比色测定法，甘油三酸酯的积累和脂解分泌的DNA，QRT-PCR的相对基因表达，分泌的蛋白质，甘油三酸酯的积累和脂解，葡萄糖和乳酸分析的能量使用。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。对激素的功能反应将评估生理反应。 AIM 3将揭示AIM 2中开发的2D和3D培养物，以胰岛素和FFA，两种刺激升高，肥胖2型糖尿病患者以及改变ECM重塑的蛋白质。将对T2DM刺激的组织反应和改变的重塑条件首先分别比较。还将使用蛋白质印迹研究已知已知的胰岛素信号传导途径的激活和炎症细胞因子的影响，以确定途径激活如何响应这些刺激和几何形状而变化。因此，提出的工作将通过开发用于发炎的脂肪组织的新3D体外模型，直接比较2D和3D培养物的结构和功能，并了解胰岛素信号对肥胖2型糖尿病患者的刺激造成损害，并与2D和3D模型中的刺激变化，并与更换的Matrix RemodeLing一起变化，从而提高了对脂肪组织工程的理解，从而直接比较了2D和3D培养物的结构和功能。