Soluble microneedles (MNs) have recently become an efficient and minimally invasive tool in transdermal drug delivery because of their excellent biocompatibility and rapid dissolution. However, direct monitoring of structural and functional changes of MNs in vivo to estimate the efficiency of insulin delivery is difficult. We monitored the dissolution of MNs to obtain structural imaging of MNs' changes by using optical coherence tomography (OCT). We also observed the effect of MNs on microvascular conditions with laser speckle contrast imaging (LSCI) and measured the blood perfusion of skin to obtain functional imaging of MNs. We determined the performance of two soluble MN arrays made from polyvinyl alcohol (PVA) and polyvinyl alcohol/ polyvinylpyrolidone (PVA/PVP) by calculating the cross-sectional areas of the microchannels in mouse skin as a function of time. Moreover, the change in blood glucose before and after using MNs loaded with insulin was evaluated as an auxiliary means to demonstrate the ability of the soluble MNs to deliver insulin. Results showed that the structural imaging of these MNs could be observed in vivo via OCT in real time and the functional imaging of MNs could be showed using LSCI. OCT and LSCI are potential tools in monitoring MNs structural and functional changes.

采用紫外-可见光谱、 荧光光谱和傅里叶变换衰减全反射红外光谱等技术, 研究在模拟人体生理Ph值条件下, 丹参酚酸A(或丹参酚酸B)与胰岛素分子之间的结合作用, 以及丹参酚酸A(或丹参酚酸B)对胰岛素二级结构的影响, 并考察葡萄糖对它们的影响。 实验结果表明, 丹参酚酸A和丹参酚酸B均能导致胰岛素内源性荧光静态猝灭; 同步荧光和三维荧光谱图表明胰岛素与丹参酚酸A(或丹参酚酸B)结合后, 构象没有发生明显变化。 红外光谱研究表明, 胰岛素与丹参酚酸A(或丹参酚酸B)结合后二级结构发生了一些改变, β-转角和无规则卷曲的相对含量略有增加, α-螺旋和β-折叠没有发生明显改变。 葡萄糖的加入会改变丹参酚酸A(或丹参酚酸B)与胰岛素的结合程度, 并加剧胰岛素构象变化以及二级结构中α-螺旋相对含量改变, 从而影响丹参酚酸A(或丹参酚酸B)-胰岛素体系中胰岛素的生物活性。

蛋白质结构失稳后会发生错误折叠、 集聚或纤维化, 不仅丧失正常功能, 有时甚至会产生生物毒性, 导致相关疾病。 纤维化了的蛋白质是神经退行性疾病以及二型糖尿病等的主要诱因, 它在溶液中具有和淀粉类似的特性, 因此蛋白质的纤维化又被称为淀粉样变性。 电磁辐射是引起蛋白质结构失稳的一个常见因素。 经电场辐照后, 蛋白质会发生去折叠和集聚, 改变自发折叠机制。 同时, 经受电场辐照的扰动而失稳的蛋白质, 纤维化过程也会受到一定的影响。 以胰岛素为研究对象, 利用硫磺素T(ThT)染色的荧光光谱法、 透射电子显微镜(TEM)以及圆二色谱(CD)法, 分别从纤维化的宏观动力学过程、 成熟纤维丝微观形貌以及原纤维二级结构组成等不同角度, 探究经33 Hz脉冲电场(PEF)不同电场强度及不同持续时间辐照后蛋白质在体外淀粉样变性机制的变化情况。 结果表明, 胰岛素溶液经过33 Hz PEF辐照后, 淀粉样变性初期产生前体蛋白的成核期延长, 原纤维丝聚集延长过程(即中间产物的寿命)缩短, 形成的成熟纤维丝长度变短且整齐成簇无分支。 这些效应随着辐照电场强度和辐照时间的增加而有不同程度的加强。 研究结果一致说明PEF辐照对胰岛素的淀粉样变性有一定的抑制作用。

Insulin resistance is a hallmark of the metabolic syndrome and type 2 diabetes. Dysfunction of PI-3K/Akt signaling was involved in insulin resistance. Glucose transporter 4 (GLUT4) is a key factor for glucose uptake in muscle and adipose tissues, which is closely regulated by PI-3K/Akt signaling in response to insulin treatment. Low-power laser irradiation (LPLI) has been shown to regulate various physiological processes and induce the synthesis or release of multiple molecules such as growth factors, which (especially red and near infrared light) is mainly through the activation of mitochondrial respiratory chain and the initiation of intracellular signaling pathways. Nevertheless, it is unclear whether LPLI could promote glucose uptake through activation of PI-3K/Akt/GLUT4 signaling in 3T3L-1 adipocytes. In this study, we investigated how LPLI promoted glucose uptake through activation of PI-3K/Akt/GLUT4 signaling pathway. Here, we showed that GLUT4 was localized to the Golgi apparatus and translocated from cytoplasm to cytomembrane upon LPLI treatment in 3T3L-1 adipocytes, which enhanced glucose uptake. Moreover, we found that glucose uptake was mediated by the PI3-K/Akt2 signaling, but not Akt1 upon LPLI treatment with Akt isoforms gene silence and PI3-K/Akt inhibitors. Collectively, our results indicate that PI3-K/Akt2/GLUT4 signaling act as the key regulators for improvement of glucose uptake under LPLI treatment in 3T3L-1 adipocytes. More importantly, our findings suggest that activation of PI3-K/Akt2/GLUT4 signaling by LPLI may provide guidance in practical applications for promotion of glucose uptake in insulin-resistant adipose tissue.

Insulin secretion is a complex and highly regulated process. Although much progress has been made in understanding the cellular mechanisms of insulin secretion and regulation, it remains unclear how conclusions from these studies apply to living animals. That few studies have been done to address these issues is largely due to the lack of suitable tools in detecting secretory events at high spatial and temporal resolution in vivo. When combined with genetically encoded biosensor, optical imaging is a powerful tool for visualization of molecular events in vivo. In this study, we generated a DNA construct encoding a secretory granule resident protein that is linked with two spectrally separate fluorescent proteins, a highly pH-sensitive green pHluorin on the intra-granular side and a red mCherry in the cytosol. Upon exocytosis of secretory granules, the dim pHluorin inside the acidic secretory granules became highly fluorescent outside the cells at neutral pH, while mCherry fluorescence remained constant in the process, thus allowing ratiometric quantification of insulin secretory events. Furthermore, mCherry fluorescence enabled tracking the movement of secretory granules in living cells. We validated this approach in insulin-secreting cells, and generated a transgenic mouse line expressing the optical sensor specifically in pancreatic β-cells. The transgenic mice will be a useful tool for future investigations of molecular mechanism of insulin secretion in vitro and in vivo.