Islet Endothelial Dysfunction in Diabetes

摘要 2型糖尿病影响了9%的美国人口。β细胞分泌功能和生存能力的受损是高血糖发生的关键。β细胞功能障碍和死亡的机制仍不完全清楚。提高我们对这些的了解对于开发更好的治疗这种衰弱疾病的方法至关重要。胰岛细胞外基质主要由胰岛内皮细胞合成，是影响β细胞的重要信号来源；细胞外基质成分层粘连蛋白和透明质酸通常促进胰岛素释放和/或β细胞存活。我们在人类2型糖尿病和动物模型的胰岛中的初步数据首次显示，层粘连蛋白的产生减少，透明质酸被破坏/碎片化，能够降解或破坏层粘连蛋白和透明质酸稳定的酶的表达增加。我们的初步数据还表明，通过在高糖中培养原代胰岛内皮细胞，可以重现这些发现。此外，我们可以通过在这种“糖尿病”的细胞外基质上培养β细胞或在层粘连蛋白β4缺乏或透明质酸碎裂的条件下培养它们来复制α细胞功能障碍。因此，我们假设在2型糖尿病中，胰岛内皮细胞来源的细胞外基质中层粘连蛋白的产生和/或信号的减少以及透明质酸片段的增加损害了胰岛β细胞的分泌功能和生存。我们的研究将包括以下三个特定目标：特定目标1：确定层粘连蛋白α4信号的过度表达/抑制是否促进/损害胰岛素的释放。我们将利用病毒的过度表达来确定“糖尿病”胰岛内皮细胞在细胞外基质中恢复正常的层粘连蛋白α4水平是否能改善胰岛素释放和/或β细胞在体外的存活。我们还将在成年、非糖尿病小鼠的α细胞中敲除层粘连蛋白受体亚单位整合素β6，并评估体内胰岛素的释放。特定目的2：确定胰岛细胞外基质中透明质酸碎裂导致胰岛素释放和β细胞存活受损的机制(S)。碎片化的透明质酸已经在非 在这里，我们将确定胰岛细胞外基质中碎片化的透明质酸是否通过TLR2/4和NFκB的激活而损害β细胞的功能和生存。具体目的3.确定破坏透明质酸(ADAMTS)和降解层粘连蛋白(MMPs)的酶的上调是否在糖尿病发病之前。使用RNA-Seq，我们将确定在UCD-T2 DM大鼠中，ADAMTS和MMP家族成员的胰岛内皮表达增加是否在糖尿病之前和/或随着糖尿病病程的延长而增加，以及在T2D人类胰岛中是否增加。我们将验证观察到的蛋白和活性水平的变化，并调节这些酶在胰岛内皮源性细胞外基质中的表达，以评估它们对胰岛素释放和β细胞存活的影响。

ABSTRACT Type 2 diabetes affects 9% of the United States population. Impairments in β-cell secretory function and survival are critical for the development of hyperglycemia. The mechanisms underlying β-cell dysfunction and death remain incompletely understood. Improving our knowledge of these is critical for the development of better therapies for this debilitating disease. The islet extracellular matrix (ECM), synthesized predominantly by islet endothelial cells, is a critical source of signals that impinge on the β-cell; ECM components laminin and hyaluronan normally enhance insulin release and/or β-cell survival. Our preliminary data in islets from human type 2 diabetes and animal models show for the first time that laminin production is decreased, hyaluronan is disrupted/fragmented, and expression of enzymes that can degrade or destabilize laminin and hyaluronan is increased. Our preliminary data also show these findings can be reproduced by culturing primary islet endothelial cells in high glucose. Further, we can reproduce β-cell dysfunction by culturing β-cells on this “diabetic” ECM or culturing them under conditions of laminin α4-deficiency or hyaluronan fragmentation. Thus, we hypothesize that in type 2 diabetes, reduced laminin production and/or signaling and increased hyaluronan fragmentation in islet endothelial-derived ECM impairs islet β-cell secretory function and survival. Our studies will encompass the following three specific aims: Specific Aim 1: To determine whether overexpression/inhibition of laminin α4 signaling improves/impairs insulin release. We will use viral overexpression to determine if restoration of normal laminin α4 levels in ECM from “diabetic” islet endothelial cells improves insulin release and/or β-cell survival in vitro. We will also knock out the laminin receptor subunit integrin α6 in β cells of adult, non-diabetic mice and assess insulin release in vivo. Specific Aim 2: To determine the mechanism(s) by which hyaluronan fragmentation in the islet ECM contributes to impaired insulin release and β-cell survival. Fragmented hyaluronan has been shown, in non- islet cells, to be proinflammatory, activating toll-like receptors (TLR) and NFκB. Here, we will determine if fragmented hyaluronan in islet ECM impairs β-cell function and survival via TLR2/4 and NFκB activation. Specific Aim 3. To determine whether upregulation of enzymes that destabilize hyaluronan (ADAMTSs) and degrade laminin (MMPs) precedes the onset of diabetes. Using RNA-Seq, we will determine if increases in islet endothelial expression of ADAMTS and MMP family members precede diabetes and/or increase with diabetes duration in UCD-T2DM rats and are increased in T2D human islets. We will validate observed changes at the protein and activity levels, and modulate expression of these enzymes in islet endothelial-derived ECM to evaluate their effects on insulin release and β-cell survival.