目标/假设
糖尿病与胰岛素分泌受损有关，通常因胰高血糖素分泌过多而加剧。理想情况下，治疗干预措施应该纠正这两种缺陷。胰高血糖素样肽 1 (GLP-1) 具有这种能力，但其究竟如何发挥其降血糖作用仍不清楚。释放后，GLP-1 迅速从 GLP-1(7–36) 降解为 GLP-1(9–36)。我们假设代谢物 GLP-1(9–36)（以前被认为没有生物活性）对胰高血糖素分泌产生直接抑制作用，并且这种机制在糖尿病中会受到损害。
方法
我们结合使用了小鼠和人类胰岛（包括来自 2 型糖尿病供体的胰岛）的胰高血糖素分泌测量、分泌颗粒动力学的全内反射荧光显微镜成像、细胞质 Ca 2+记录和蛋白激酶 A 活性测量、免疫细胞化学、体内生理学和GTP结合蛋白解离研究，探讨GLP-1如何发挥其对胰高血糖素分泌的抑制作用以及代谢物GLP-1的作用(9–36)。
结果
GLP-1(7–36) 抑制分离胰岛中的胰高血糖素分泌，IC 50为 2.5 pmol/l。在低葡萄糖浓度下效果尤其强烈。降解产物 GLP-1(9-36) 也具有这种能力。 GLP-1(9–36) 在 GLP-1 受体遗传/药理学失活后保留其胰高血糖素作用。 GLP-1(9–36) 还可有效抑制 β-肾上腺素能刺激、氨基酸和膜去极化引起的胰高血糖素分泌。在胰岛 α 细胞中，GLP-1(9–36) 导致通过对 ω-琼脂毒素敏感的电压门控 Ca 2+通道抑制 Ca 2+进入，从而导致分泌颗粒对接池的百日咳毒素敏感性耗尽，胰高血糖素受体拮抗剂 REMD2.59 和 L-168049 可阻止的作用。患有 2 型糖尿病的人类捐赠者的 α 细胞中，GLP-1(9–36) 抑制胰高血糖素分泌和减少对接颗粒数量的能力丧失了。在体内，高外源浓度的 GLP-1(9–36) (>100 pmol/l) 导致胰岛素诱导的低血糖期间循环胰高血糖素小幅降低 (30%)。 REMD2.59 消除了这种效应，它立即使循环胰高血糖素增加>225%（根据血浆葡萄糖的变化进行调整），而不影响胰腺胰高血糖素含量。
结论/解释
我们得出结论，GLP-1 代谢物 GLP-1(9-36) 是胰高血糖素分泌的全身抑制剂。我们提出，在小鼠和 2 型糖尿病患者中，胰高血糖素信号传导基因/药理学失活后观察到的循环胰高血糖素增加反映了 GLP-1(9-36) 的胰高血糖素抑制作用的消除。
图解摘要
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GLP-1 metabolite GLP-1(9–36) is a systemic inhibitor of mouse and human pancreatic islet glucagon secretion
Aims/hypothesis
Diabetes mellitus is associated with impaired insulin secretion, often aggravated by oversecretion of glucagon. Therapeutic interventions should ideally correct both defects. Glucagon-like peptide 1 (GLP-1) has this capability but exactly how it exerts its glucagonostatic effect remains obscure. Following its release GLP-1 is rapidly degraded from GLP-1(7–36) to GLP-1(9–36). We hypothesised that the metabolite GLP-1(9–36) (previously believed to be biologically inactive) exerts a direct inhibitory effect on glucagon secretion and that this mechanism becomes impaired in diabetes.
Methods
We used a combination of glucagon secretion measurements in mouse and human islets (including islets from donors with type 2 diabetes), total internal reflection fluorescence microscopy imaging of secretory granule dynamics, recordings of cytoplasmic Ca2+ and measurements of protein kinase A activity, immunocytochemistry, in vivo physiology and GTP-binding protein dissociation studies to explore how GLP-1 exerts its inhibitory effect on glucagon secretion and the role of the metabolite GLP-1(9–36).
Results
GLP-1(7–36) inhibited glucagon secretion in isolated islets with an IC50 of 2.5 pmol/l. The effect was particularly strong at low glucose concentrations. The degradation product GLP-1(9–36) shared this capacity. GLP-1(9–36) retained its glucagonostatic effects after genetic/pharmacological inactivation of the GLP-1 receptor. GLP-1(9–36) also potently inhibited glucagon secretion evoked by β-adrenergic stimulation, amino acids and membrane depolarisation. In islet alpha cells, GLP-1(9–36) led to inhibition of Ca2+ entry via voltage-gated Ca2+ channels sensitive to ω-agatoxin, with consequential pertussis-toxin-sensitive depletion of the docked pool of secretory granules, effects that were prevented by the glucagon receptor antagonists REMD2.59 and L-168049. The capacity of GLP-1(9–36) to inhibit glucagon secretion and reduce the number of docked granules was lost in alpha cells from human donors with type 2 diabetes. In vivo, high exogenous concentrations of GLP-1(9–36) (>100 pmol/l) resulted in a small (30%) lowering of circulating glucagon during insulin-induced hypoglycaemia. This effect was abolished by REMD2.59, which promptly increased circulating glucagon by >225% (adjusted for the change in plasma glucose) without affecting pancreatic glucagon content.
Conclusions/interpretation
We conclude that the GLP-1 metabolite GLP-1(9–36) is a systemic inhibitor of glucagon secretion. We propose that the increase in circulating glucagon observed following genetic/pharmacological inactivation of glucagon signalling in mice and in people with type 2 diabetes reflects the removal of GLP-1(9–36)’s glucagonostatic action.
Graphical Abstract
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