Blockade of glucagon receptor induces alpha-cell hypersecretion by hyperaminoacidemia in mice

Blockade of the glucagon receptor (GCGR) has been shown to improve glycemic control. However, this therapeutic approach also brings side effects, such as alpha-cell hyperplasia and hyperglucagonemia, and the mechanisms underlying these side effects remain elusive. Here, we conduct single-cell transcriptomic sequencing of islets from male GCGR knockout (GCGR-KO) mice. Our analysis confirms the elevated expression of Gcg in GCGR-KO mice, along with enhanced glucagon secretion at single-cell level. Notably, Vgf (nerve growth factor inducible) is specifically upregulated in cells of GCGR-KO mice. Inhibition of VGF impairs the formation of glucagon immature secretory granules and compromises glucagon maturation, lead to reduced -cell hypersecretion of glucagon. We further demonstrate that activation of both mTOR-STAT3 and ERK-CREB pathways, induced by elevated circulation amino acids, is responsible for upregulation of Vgf and Gcg expression following glucagon receptor blockade. Thus, our findings elucidate a previously unappreciated molecular mechanism underlying hyperglucagonemia in GCGR blockade. The pancreatic islets were isolated from 8-12-week-old male C57BL/6J mice. To prepare the single-cell suspension, the purified islets were digested with 0.25% trypsin-EDTA and filtered through a 40 m cell strainer. Cell viability was evaluated by trypan blue staining, followed by microscopy to ensure that cell viability was greater than 90%, and over 90% of cells were singlets.胰高血糖素受体（glucagon receptor, GCGR）阻断已被证实可改善血糖控制。然而，该治疗策略同时会引发α细胞增生、高胰高血糖素血症等不良反应，其潜在分子机制仍尚不明确。本研究对雄性胰高血糖素受体敲除（GCGR-KO）小鼠的胰岛开展单细胞转录组测序。分析结果证实，GCGR-KO小鼠体内Gcg的表达水平显著升高，且单细胞层面的胰高血糖素分泌功能增强。值得注意的是，神经生长因子诱导蛋白（nerve growth factor inducible, Vgf）在GCGR-KO小鼠的α细胞中特异性上调。抑制Vgf的表达会破坏胰高血糖素未成熟分泌颗粒的形成，损害胰高血糖素的成熟过程，进而降低α细胞的胰高血糖素过度分泌水平。本研究进一步证实，循环氨基酸水平升高所激活的mTOR-STAT3与ERK-CREB两条通路，共同介导了胰高血糖素受体阻断后Vgf与Gcg的表达上调。综上，本研究阐明了胰高血糖素受体阻断后高胰高血糖素血症背后一种此前未被认知的分子机制。 本研究从8~12周龄的雄性C57BL/6J小鼠体内分离胰岛。为制备单细胞悬液，将纯化后的胰岛用0.25%胰蛋白酶-乙二胺四乙酸（trypsin-EDTA）消化，并通过40 μm细胞筛过滤。采用台盼蓝染色评估细胞活力，辅以显微镜观测以确保细胞活力高于90%，且超过90%的细胞为单个游离细胞。