Single Cell Peptide Heterogeneity of Rat Islets of Langerhans

Abstract

Measuring the chemical composition of individual cells in mammalian organs can provide critical insights toward understanding the mechanisms leading to their normal and pathological function. In this work, single cell heterogeneity of islets of Langerhans is characterized with high throughput by microscopy-guided single cell matrix-assisted laser desorption/ionization mass spectrometry. Two levels of chemical heterogeneity were observed from the analysis of more than 3000 individual cells. Within a single islet, cellular heterogeneity was evident from the exclusive expression of the canonical biomarkers glucagon, insulin, pancreatic polypeptide (PP), and somatostatin within α-, β-, γ-, and δ-cells, respectively. We localized the neuropeptide WE-14, a known cell-to-cell signaling molecule, to individual δ-cells. Moreover, several unreported endogenous peptides generated by dibasic site cleavages of PP were detected within individual γ-cells. Of these, PP(27-36) was previously shown to activate the human Y4 receptor, suggesting it has a signaling role in vivo. Heterogeneity in cell composition was also observed between islets as evidenced by a 50-fold larger α-cell population in islets of the dorsal pancreas compared to the ventral-derived pancreatic islets. Finally, PP(27-36) was more abundant in γ-cells from the ventral region of the pancreas, indicating differences in the extent of PP-prohormone processing in the two regions of the pancreas.

Figure 1

Representative mass spectra acquired from individual α-, β-, γ-, and δ-cells, as well as an intact islet of Langerhans collected from the dorsal pancreas. Peptide cell-type qualifiers/biomarkers: glucagon m/z 3481.5 (α-cells), insulin m/z 5799.9 (β-cells), PP m/z 4397.2 (γ-cells), and somatostatin-14 m/z 1637.7 (δ-cells). These signals are highlighted with asterisks. Many other characteristic signals were observed in the cells as described herein.

Figure 2

MALDI MS characterization of intact islets of Langerhans and corresponding single cell populations obtained from individual islets. (a) Intact islets have different peptide profiles in the dorsal (filled circles) and ventral (crosses) pancreas. Distributions of total ion current (TIC)-normalized intensities of insulin, glucagon, and PP signals acquired from intact islets. (b and c) Results of k-means clustering of single cell data shown in color: α-cells (red), β-cells (green), γ-cells (blue), δ-cells (purple). K-means clustering was used on the full data set to classify and count the different cell types observed in single cell preparations from islets of Langerhans based on TIC-normalized peptide signal intensities. The classification highlights differences in cell composition between islets from dorsal and ventral pancreata, reflected in the relative abundance of α- (red) and γ- (blue) cells. The data were dimension-reduced with principal component analysis for visualization purposes only, here shown with principal components (PC) 1, 2, and 4. (b) Dorsal pancreas islet cells (n = 1768). (c) Ventral pancreas islet cells (n = 1738).

Figure 3

Sequence and predicted structure of peptides originating from the rat pancreatic prohormone. (a) Sequence of rat pancreatic prohormone (UNIPROT ID: P06303, PAHO_RAT); the dibasic sites are underlined. (b) Observed peptide products resulting from processing of the pancreatic prohormone (filled color boxes). (c) Ab initio-calculated structure of PP; the dibasic site is outlined in magenta. (d) Molecular modeling shows that the N- and C-terminal products (sea green and sky blue, respectively) resulting from cleavage at the dibasic site retain the α-helix folding of the parent peptide.

Figure 4

Comparison of pancreatic prohormone peptide signals acquired from γ-cells of the dorsal and ventral islets (n_Dγ = 79 cells, n_Vγ = 418 cells). Box and whisker plots of TIC-normalized signal intensities are shown for (a) peptide products from internal dibasic cleavage of PP, left: PP(1–24), m/z 2808.2; right: PP(27–36), m/z 1295.7. (b) Peptide products from internal monobasic cleavage of PP, left: PP(1–16), m/z 1818.8; right: PP(18–36), m/z 2441.3. (c) Canonical peptide products from the pancreatic prohormone, left: C-terminal peptide, m/z 3037.4; right: PP, m/z 4397.2.

Figure 5

Comparison of mass spectra acquired from a single γ-cell (top) and a pixel on a pancreatic islet cross-section (bottom). Color-coded molecular ion distributions for four peptides are shown on the MALDI MS image (center).