The leptin receptor has no role in delta-cell control of beta-cell function in the mouse

Introduction: Leptin inhibits insulin secretion from isolated islets from multiple species, but the cell type that mediates this process remains elusive. Several mouse models have been used to explore this question. Ablation of the leptin receptor (Lepr) throughout the pancreatic epithelium results in altered glucose homeostasis and ex vivo insulin secretion and Ca2+ dynamics. However, Lepr removal from neither alpha nor beta cells mimics this result. Moreover, scRNAseq data has revealed an enrichment of LEPR in human islet delta cells. Methods: We confirmed LEPR upregulation in human delta cells by performing RNAseq on fixed, sorted beta and delta cells. We then used a mouse model to test whether delta cells mediate the diminished glucose-stimulated insulin secretion in response to leptin. Results: Ablation of Lepr within mouse delta cells did not change glucose homeostasis or insulin secretion, whether mice were fed a chow or high-fat diet. We further show, using a publicly available scRNAseq dataset, that islet cells expressing Lepr lie within endothelial cell clusters. Conclusions: In mice, leptin does not influence beta-cell function through delta cells. We aimed to understand the differences in expression between human beta and delta cells. To complete this project, we obtained islet samples from five non-diabetic human donors. Current protocols for sorting live islets do not reliably distinguish beta and delta cells. Therefore, we used a procedure to sort cells using internal cell-specific markers. Islets were fixed and suspended into a single-cell suspension. The cell membranes were perforated, and cells were indirectly fluorescently immunolabeled using antibodies against insulin and somatostatin. We found that delta cells express the machinery that beta cells use to sense glucose and secrete insulin (except for insulin). Delta cells were also enriched for genes such as Somatostatin, HHex, Leptin Receptor, Erb-B2 Receptor Tyrosine Kinase 4 and Dopamine Receptor 2, Serpin Family A Member 1, Nuclear receptor coactivator 7, several GABA receptors, Growth Hormone Receptor, and several solute transporters. Characterization of differentially expressed cell-surface molecules could lead to better sorting of live human beta and delta cells.Islets from non-diabetic human donors were obtained from the IIDP, cultured in Prodo Complete Media, then fixed in 1% PFA, 01.% saponin, and 1:100 Rnasin before staining with Somatostatin and Insulin and FACS sorting

引言：瘦素（Leptin）可抑制多种物种分离胰岛的胰岛素分泌，但介导该过程的细胞类型仍未明确。已有多项小鼠模型被用于探究此问题。在胰腺上皮细胞中全面敲除瘦素受体（leptin receptor, Lepr）会导致葡萄糖稳态异常、离体胰岛素分泌及钙（Ca²+）动力学改变。然而，仅在α细胞或β细胞中敲除Lepr并不能复刻这一表型。此外，单细胞RNA测序（scRNAseq）数据显示，人类胰岛δ细胞中LEPR的表达存在富集。
方法：我们通过对固定、分选后的β细胞与δ细胞进行RNA测序（RNAseq），验证了人类δ细胞中LEPR的表达上调。随后我们利用小鼠模型，验证δ细胞是否介导了瘦素诱导的葡萄糖刺激胰岛素分泌减弱效应。
结果：无论小鼠喂食普通饲料还是高脂饲料，在小鼠δ细胞中敲除Lepr均不会改变葡萄糖稳态或胰岛素分泌。此外，通过公开的scRNAseq数据集，我们进一步证实表达Lepr的胰岛细胞属于内皮细胞簇。
结论：在小鼠体内，瘦素并不会通过δ细胞影响β细胞功能。本研究旨在探究人类β细胞与δ细胞之间的表达差异。为完成本项目，我们从5名非糖尿病人类供体中获取了胰岛样本。当前的活胰岛分选方案无法可靠区分β细胞与δ细胞，因此我们采用基于细胞内特异性标志物的分选方法：将胰岛固定并制备为单细胞悬液，穿孔细胞膜后，使用针对胰岛素与生长抑素的抗体进行间接荧光免疫标记。我们发现，δ细胞表达β细胞用于感知葡萄糖并分泌胰岛素的全套分子机制（胰岛素本身除外）。δ细胞还富集了诸如生长抑素（Somatostatin）、Hhex、瘦素受体（Leptin Receptor）、Erb-B2受体酪氨酸激酶4（Erb-B2 Receptor Tyrosine Kinase 4）、多巴胺受体2（Dopamine Receptor 2）、丝氨酸蛋白酶抑制剂家族A成员1（Serpin Family A Member 1）、核受体辅激活因子7（Nuclear receptor coactivator 7）、多种GABA受体、生长激素受体（Growth Hormone Receptor）以及多种溶质转运蛋白相关基因。对差异表达的细胞表面分子进行表征，可实现更高效的活人β细胞与δ细胞分选。本研究中的非糖尿病人类供体胰岛由IIDP提供，在Prodo完全培养基中培养后，使用1%多聚甲醛（PFA）、0.1%皂角苷与1:100比例的Rnasin进行固定，随后进行生长抑素、胰岛素染色与荧光激活细胞分选（FACS）。