Single-Cell Transcriptional Profiling of Mouse Islets Following Short-Term ObesogenicDietary Intervention. Single-Cell Transcriptional Profiling of Mouse Islets Following Short-Term ObesogenicDietary Intervention

Obesity is closely associated with adipose tissue inflammation and insulin resistance. Dysglycemia and type 2 diabetes results when islet β cells fail to maintain appropriate insulin secretion in the face of insulin resistance. To clarify the early transcriptional events leading to β-cell failure in the setting of obesity, we fed male C57BL/6J mice an obesogenic, high fat diet (60% kcal from fat) or control diet (10% kcal from fat) for one week and islets from these mice (from 4 high fat- and 3 control-fed mice) were subjected to single cell RNA sequencing analysis. Islet endocrine cell types (α cells, β cells, δ cells, PP cells) and other resident cell types (macrophages, T cells) were annotated by transcript profiles and visualized using Uniform Manifold Approximation and Projection for Dimension Reduction (UMAP) plots. UMAP analysis revealed distinct cell sub-populations (11 for β cells, 5 for α cells, 3 for δ cells, PP cells, ductal cells, endothelial cells), emphasizing the heterogeneity of cell populations in the islet. We identified that distinct β cell populations downregulate genes associated with the endoplasmic reticulum stress response and upregulate genes associated with insulin secretion, while others upregulate genes that impair insulin secretion, cellular proliferation, and survival. Moreover, all β cell populations negatively regulate genes associated with immune response activation. Our data indicate that an early transcriptional response in islets to an obesogenic diet reflects an attempt by distinct populations of β cells to augment or impair cellular function, possible harbingers of ensuing insulin resistance. Overall design: Islet isolation. Male C57BL/6J mouse pancreas were isolated from 8 weeks of age, animals were fed either a LFD (10% kcal from fat; Research Diets D12450B), or HFD (60% kcal from fat; Research Diets D12492) for one week. Islets were isolated from collagenase-perfused pancreata as previously described (Stull, 2017). After isolation, mouse islets were handpicked and digested with Accutase (EMD Millipore Corporation) containing 2U/ml of DNAse during 5min at 37°C sob agitation (1000rpm). Digested cell islets were washed several times with PBS+2%FBS to eliminate DNAse and then filtered using a cell strainer (40mm). Single cells suspension and samples with more than 90% viability were used for scRNAseq.

肥胖与脂肪组织炎症及胰岛素抵抗密切相关。当胰岛β细胞无法在胰岛素抵抗状态下维持正常胰岛素分泌时，便会引发糖代谢异常与2型糖尿病。为阐明肥胖背景下导致β细胞功能衰竭的早期转录事件，我们将雄性C57BL/6J小鼠分别饲喂致肥胖性高脂饲料（脂肪供能占比60%）或对照饲料（脂肪供能占比10%），持续1周；随后从4只高脂饲料饲喂小鼠与3只对照饲料饲喂小鼠中分离胰岛，进行单细胞RNA测序（single cell RNA sequencing, scRNAseq）分析。胰岛内分泌细胞类型（α细胞、β细胞、δ细胞、PP细胞）及其他驻留细胞类型（巨噬细胞、T细胞）通过转录谱进行注释，并使用均匀流形近似与投影降维（Uniform Manifold Approximation and Projection for Dimension Reduction, UMAP）图进行可视化。UMAP分析揭示了独特的细胞亚群：β细胞包含11个亚群，α细胞包含5个亚群，δ细胞、PP细胞、导管细胞及内皮细胞各包含3个亚群，这凸显了胰岛细胞群体的异质性。研究发现，不同的β细胞亚群分别下调内质网应激反应相关基因并上调胰岛素分泌相关基因，而另有亚群则上调会损害胰岛素分泌、细胞增殖与存活的基因。此外，所有β细胞亚群均负向调控免疫应答激活相关基因。我们的数据表明，胰岛在高脂致肥胖饲料刺激下的早期转录反应，反映了不同β细胞亚群增强或损害细胞功能的尝试，这可能是后续胰岛素抵抗的先兆。总体实验设计：胰岛分离。从8周龄雄性C57BL/6J小鼠体内分离胰腺，小鼠分别饲喂低脂饲料（low fat diet, LFD，脂肪供能占比10%；Research Diets公司货号D12450B）或高脂饲料（high fat diet, HFD，脂肪供能占比60%；Research Diets公司货号D12492），持续1周。按照此前报道的方法（Stull, 2017），从胶原酶灌注的胰腺中分离胰岛。分离完成后，手动挑选小鼠胰岛，在37℃、1000rpm振荡条件下，使用含2U/ml脱氧核糖核酸酶（DNAse）的Accutase（EMD Millipore Corporation公司产品）消化5分钟。消化后的胰岛细胞用PBS+2%胎牛血清（FBS）多次洗涤以去除脱氧核糖核酸酶，随后通过40μm细胞筛过滤。将单细胞悬液且活细胞率高于90%的样本用于单细胞RNA测序。