Before insulin can stimulate myocytes to take up glucose, it must first move from the circulation to the interstitial space. The continuous endothelium of skeletal muscle (SkM) capillaries restricts insulin’s access to myocytes. The mechanism by which insulin crosses this continuous endothelium is critical to understand insulin action and insulin resistance; however, methodological obstacles have limited understanding of endothelial insulin transport in vivo. Here, we present an intravital microscopy technique to measure the rate of insulin efflux across the endothelium of SkM capillaries. This method involves development of a fully bioactive, fluorescent insulin probe, a gastrocnemius preparation for intravital microscopy, an automated vascular segmentation algorithm, and the use of mathematical models to estimate endothelial transport parameters. We combined direct visualization of insulin efflux from SkM capillaries with modeling of insulin efflux kinetics to identify fluid-phase transport as the major mode of transendothelial insulin efflux in mice. Model-independent experiments demonstrating that insulin movement is neither saturable nor affected by insulin receptor antagonism supported this result. Our finding that insulin enters the SkM interstitium by fluid-phase transport may have implications in the pathophysiology of SkM insulin resistance as well as in the treatment of diabetes with various insulin analogs.

中文翻译：

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胰岛素通过液相运输离开骨骼肌毛细血管


在胰岛素刺激肌细胞吸收葡萄糖之前，它必须首先从循环系统转移到细胞间隙。骨骼肌 (SkM) 毛细血管的连续内皮限制胰岛素进入肌细胞。胰岛素穿过连续内皮的机制对于了解胰岛素作用和胰岛素抵抗至关重要；然而，方法学上的障碍限制了对体内内皮胰岛素转运的了解。在这里，我们提出了一种活体显微镜技术来测量胰岛素穿过 SkM 毛细血管内皮的流出率。该方法涉及开发完全生物活性的荧光胰岛素探针、用于活体显微镜检查的腓肠肌制剂、自动血管分割算法以及使用数学模型来估计内皮运输参数。我们将 SkM 毛细血管的胰岛素流出的直接可视化与胰岛素流出动力学建模相结合，以确定液相运输是小鼠经内皮胰岛素流出的主要模式。独立于模型的实验证明胰岛素运动既不可饱和也不受胰岛素受体拮抗作用的影响，支持了这一结果。我们发现胰岛素通过液相运输进入 SkM 间质，这可能对 SkM 胰岛素抵抗的病理生理学以及各种胰岛素类似物治疗糖尿病具有影响。