Do the responsive -cell clusters remain robustly responsive in their gene expression patterns with more prolonged feeding? Do the less responsive clusters become more responsive with time? Are specific clusters more susceptible to apoptosis or dedifferentiation over time? Does the islet isolation process influence mRNA expression? In future studies, applying our annotation and clustering analyses should allow us to address these questions using timed feeding cohorts

Do the responsive -cell clusters remain robustly responsive in their gene expression patterns with more prolonged feeding? Do the less responsive clusters become more responsive with time? Are specific clusters more susceptible to apoptosis or dedifferentiation over time? Does the islet isolation process influence mRNA expression? In future studies, applying our annotation and clustering analyses should allow us to address these questions using timed feeding cohorts. identified that distinct -cell clusters downregulate genes associated with the endoplasmic reticulum stress response and upregulate genes associated with insulin secretion, whereas others upregulate genes that impair insulin secretion, cell proliferation, and cell survival. Moreover, all -cell clusters negatively regulate genes associated with immune response activation. Glucagon-producing cells exhibited patterns similar to cells but, again, in clusters containing the minority of cells. Our data indicate that an early transcriptional response in islets to an obesogenic diet reflects an attempt by distinct populations of cells to augment or impair cellular function and/or reduce inflammatory responses as possible harbingers of ensuing insulin resistance. mice fed for one week with either a high-fat diet (HFD, 60% kcal from fat, = 4) or a control low-fat diet (LFD, 10% kcal from fat, = 3) and used to perform single-cell RNA sequencing. (a) Annotation of islet cell types into different clusters based on Gastrodenol the expression of key identifying genes, depicted in the Uniform Manifold Approximation and Projection for Dimension Reduction (UMAP) plots of merged sc-RNAseq profiles from LFD and HFD mice; (b) UMAP of single-cell RNA sequencing profiles from islets of individual mice fed a HFD or a LFD, as indicated; In (cCf), the percentage of cells (c), cells (d), cells (e), and other cell types (f) identified per cluster relative to the total number of cells sequenced is shown. Data are mean SEM, = 3 for LFD and above each bar indicates the average number of cells per cluster. 2.2. Single-Cell RNA Sequencing Analysis Reveals Greatest Gene Expression Changes in Minor Cell Clusters Following Short-Term HFD Feeding Next, we examined the gene expression profiles in cells from mice fed either a HFD or LFD for one week. UMAP analysis identified a total of 11 distinct -cell clusters Gastrodenol (1-11, see Figure 1a and Figure 2a). Based on the proportion of cells per cluster (Figure 1c), we identified three major clusters of cells (1C3) and eight minor clusters (4C11). Differential gene expression between HFD and LFD were interrogated, and statistical significance was determined by using edgeR on the integrated single-cell data obtained by the R package Seurat (see Section 4). It is notable that the major clusters of cells (1C3) showed minimal change in gene expression patterns (Supplementary Figure S2), whereas the greatest changes were observed in the minor clusters (most notably 5, 7, 8, 10, 11) (Figure 2b). These findings suggest that minor -cell clusters drive the earliest responses to HFD, and further emphasize how bulk RNA sequencing might miss these sentinel changes. In these minor clusters, the most notable gene Gastrodenol expression changes reflect upon hormone secretion and intracellular inflammatory pathways. Clusters 5, 7, 8, 10, and 11 demonstrated significant in genes that promote insulin secretion (genes, in this group of clusters following HFD feeding. Open in a separate window Figure 2 Identification of differentially expressed genes of the minor Gastrodenol -cell clusters. -cell clusters were identified from dissociated islets from male mice fed for one week with either a high-fat diet (HFD, 60% kcal from fat) or a control low-fat diet (LFD, 10% kcal from fat). (a) Representative UMAP plot of -cell clusters identified by single-cell RNA sequencing; (b) heatmaps of the minor -cell clusters of genes significantly differentially expressed ( 0.05) in the -cell clusters 5, 7, 8, 10, and 11; genes are ordered from most positive to most negative fold-change. Among the -cell clusters that showed minimal gene expression changes between HFD and LFD, four of them (1, 2, 6, and 9) showed downregulation of genes for the Rabbit polyclonal to PIWIL2 endoplasmic reticulum stress response (and mice fed for one week with either a high-fat diet (HFD, 60% kcal from fat) or a control low-fat diet (LFD, 10% kcal from fat). (a) Representative UMAP plot of -cell clusters; (b) heatmaps depicting.