Light-Mediated Enhancement of Glucose-Stimulated Insulin Release of Optogenetically Engineered Human Pancreatic Beta-Cells
Light-Mediated Enhancement of Glucose-Stimulated Insulin Release of Optogenetically Engineered Human Pancreatic Beta-Cells
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DOI：
10.1021/acssynbio.3c00653
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发表时间：
2024-02-20
影响因子：
4.7
通讯作者：
Tzanakakis，Emmanuel S.
中科院分区：
文献类型：
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作者：
Chen，Zijing;Stoukides，Demetrios M.;Tzanakakis，Emmanuel S.
在外源递送的胰腺β-细胞中增强葡萄糖刺激的胰岛素分泌（GSIS）是期望的，例如，以克服2型糖尿病中表现出的胰岛素抗性或减少用于支持1型糖尿病中血糖稳态的β-细胞的数量。光遗传工程细胞可以通过暴露于光来增强其功能。鉴于环腺苷酸（cAMP）介导GSIS，我们推测GSIS的光放大在携带可光活化的腺苷酸环化酶（PAC）的人β细胞中是可行的。为此，人类EndoC-βH3细胞被改造为表达蓝光激活的PAC，并建立了一个工作流程，将假胰岛（PI）的可扩展制造与有效的腺病毒转导相结合，导致超过80%的细胞携带PAC。用PAC体外光活化法测定细胞内cAMP和GSIS的变化，以及PAC包封和植入链脲佐菌素诱导的糖尿病小鼠体内后的变化。在光照下表达PAC的β细胞中cAMP迅速升高，并在光照终止后迅速下降。在升高的葡萄糖中，光诱导的cAMP扩增伴随着比没有PAC的β细胞大2倍的GSIS。增强的GSIS保留了其双相模式，耗氧率保持不变。接受工程化β细胞PI的糖尿病小鼠与保持在黑暗中或不接受细胞的那些相比在照明时表现出改善的葡萄糖耐量。这些发现支持使用光遗传学对β细胞进行分子定制，以更好地治疗糖尿病，而不会产生药理学方法的不良影响。
Enhancement of glucose-stimulated insulin secretion (GSIS) in exogenously delivered pancreatic β-cells is desirable, for example, to overcome the insulin resistance manifested in type 2 diabetes or to reduce the number of β-cells for supporting homeostasis of blood sugar in type 1 diabetes. Optogenetically engineered cells can potentiate their function with exposure to light. Given that cyclic adenosine monophosphate (cAMP) mediates GSIS, we surmised that optoamplification of GSIS is feasible in human β-cells carrying a photoactivatable adenylyl cyclase (PAC). To this end, human EndoC-βH3 cells were engineered to express a blue-light-activated PAC, and a workflow was established combining the scalable manufacturing of pseudoislets (PIs) with efficient adenoviral transduction, resulting in over 80% of cells carrying PAC. Changes in intracellular cAMP and GSIS were determined with the photoactivation of PAC in vitro as well as after encapsulation and implantation in mice with streptozotocin-induced diabetes. cAMP rapidly rose in β-cells expressing PAC with illumination and quickly declined upon its termination. Light-induced amplification in cAMP was concomitant with a greater than 2-fold GSIS vs β-cells without PAC in elevated glucose. The enhanced GSIS retained its biphasic pattern, and the rate of oxygen consumption remained unchanged. Diabetic mice receiving the engineered β-cell PIs exhibited improved glucose tolerance upon illumination compared to those kept in the dark or not receiving cells. The findings support the use of optogenetics for molecular customization of the β-cells toward better treatments for diabetes without the adverse effects of pharmacological approaches.