Adenosine kinase inhibition selectively promotes rodent and porcine islet β-cell replication

Abstract

Diabetes is a pathological condition characterized by relative insulin deficiency, persistent hyperglycemia, and, consequently, diffuse micro- and macrovascular disease. One therapeutic strategy is to amplify insulin-secretion capacity by increasing the number of the insulin-producing β cells without triggering a generalized proliferative response. Here, we present the development of a small-molecule screening platform for the identification of molecules that increase β-cell replication. Using this platform, we identify a class of compounds [adenosine kinase inhibitors (ADK-Is)] that promote replication of primary β cells in three species (mouse, rat, and pig). Furthermore, the replication effect of ADK-Is is cell type-selective: treatment of islet cell cultures with ADK-Is increases replication of β cells but not that of α cells, PP cells, or fibroblasts. Short-term in vivo treatment with an ADK-I also increases β-cell replication but not exocrine cell or hepatocyte replication. Therefore, we propose ADK inhibition as a strategy for the treatment of diabetes.

Fig. 1.

Experimental outline and rat islet culture composition. (A) Schematic outline of the screening protocol used to identify compounds that promote β-cell replication. Isolated rat islets were rested overnight before being dispersed and plated. Islet cultures were allowed 48 h to adhere before a 24-h compound treatment period. Cultures were then fixed, stained, and analyzed. (B) Composition of rat islet cultures. Left, Immunofluorescent staining of dispersed rat islet cells for the β-cell transcription factor PDX-1 (green), α-cell hormone glucagon (red), fibroblast marker vimentin (yellow), and DNA (blue) in combination. PDX-1 staining alone is also shown (Left, lower right corner). Right, Quantification (percentage total) of the populations stained in the left panel. Percentages were obtained by automated image acquisition (n = 80) and analysis from a representative rat islet preparation. The SD for all values was <10%. (C) PDX-1-positive cells (blue) express either insulin (green) or somatostatin (red). (Scale bar: 100 μm.)

Fig. 2.

ADK-Is induce proliferation of rat, mouse, and porcine β cells. (A) Representative images of DMSO- and 5-IT-treated islet cell cultures; β cells (PDX-1; green), replicating cells (ki-67; red), and replicating β cells (merged; yellow). Replicating β cells (yellow arrows) and replicating non-β cells (white arrows) are identified. (B) Dose–response curves for rat islet cultures showing the relationship between β-cell proliferation and compound treatment with 5-IT (Left; EC₅₀ = 4.7 μM) and ABT-702 (Right; EC₅₀ = 7.0 μM). (C) Quantification of β-cell number after treatment with DMSO or 5-IT (2 μM) for 96 and 144 h. Error bars represent SD; *P < 0.01 compared with the vehicle treatment condition. See SI Methods for experimental details of cell quantification.

Fig. 3.

β cells express nuclear ADK, which acts as a cell-autonomous negative regulator of proliferation. (A–C) Nuclear ADK staining (red) is present in β cells (insulin; green) (A) but not α cells (glucagon, green) (B) or fibroblasts (vimentin; green) (C). (D) Somatostatin cells (green) demonstrate variable intensities of nuclear ADK expression. White arrows identify representative cells in each image. (Scale bars: 100 μm.) (E) Quantification of β-cell replication after infection with virus containing either a scrambled RNAi sequence (Left) or an ADK-directed RNAi sequence (Right). The replication rate of uninfected cells (blue) and infected cells (red) within the same well were analyzed separately on the basis of virus-encoded GFP expression. Error bars represent SEM (n = 8 independent wells); *P < 0.01. See SI Methods for additional experimental details.

Fig. 4.

Induction of β-cell replication by ADK-Is is mTOR pathway-dependant and additive to glucose and glucagon-like peptide 1 receptor (GLP-1R) agonists. (A) PDX-1⁺-cell replication rates were quantified in the presence of DMSO only, 5-IT plus DMSO, and 5-IT plus the small molecule inhibitor indicated below each bar. Data are shown as the fold increases above the DMSO-only treatment condition. The inhibitors were used at a concentration of 10 μM (*P < 0.05 compared with the DMSO plus 5-IT treatment condition). (B) Cell lysates were collected from the rat INS-1E β cell line after a 15-min treatment with the indicated compound. Western blot was performed by serially probing for phosphorylated RP-S6 and for total RP-S6 protein (loading control). See SI Methods for additional experimental details. (C) Quantification of the β-cell replication rate after culture for 24, 48, or 96 h in the presence of various glucose concentrations plus DMSO or 5-IT (2 μM). Values are normalized to the 5 mM glucose plus DMSO treatment condition at each time point. The SD was <10% for each treatment condition. Error bars are not shown; *P < 0.01 when 5-IT treated wells are compared with DMSO treated wells at the same glucose concentration and time point; **P < 0.01 when DMSO or 5-IT treated wells are compared with the 5 mM glucose-treated condition at the same time point and with same treatment (DMSO or 5-IT). (D) Quantification of the β-cell replication rate after treatment with DMSO, 5-IT, Ex4 (exendin 4), GLP-1, 5-IT plus Ex4, and 5-IT plus GLP-1 for 24 h. The concentration of 5-IT was 2 μM. The concentrations of Ex4 and GLP-1 were 20 nM (light colors; Left) and 4 nM (dark colors; Right). Values normalized to the DMSO treatment condition. Error bars represent SD; *P < 0.01 and **P < 0.03 for the indicated comparisons.

Fig. 5.

ADK-Is selectively promote β-cell replication in vitro and in vivo. (A) Representative images used to analyze the replication rates of PP cells (pancreatic peptide), α cells (glucagon), δ cells (somatostatin), and fibroblasts (vimentin) present in the in vitro rat islet culture. The molecular marker of cell identity is red, ki-67 is green, and nuclear staining is blue. The red box within the glucagon-stained image (Top Right) is a high magnification view of two ki-67 and glucagon double positive cells (arrow). (B) Quantification of the in vitro replication rates of δ cells (somatostatin⁺; Top Left), α cells (glucagon⁺; Top Right), and fibroblasts (vimentin⁺; Bottom Left) after treatment with DMSO, 5-IT (2 μM), or ABT-702 (15 μM). *P < 0.01 and **P < 0.05 compared with DMSO-treated condition. (C) Quantification of the replication rate of isolated murine hepatocytes grown in the presence of EGF (40 ng/mL) and HGF (20 ng/mL) plus DMSO or ABT-702 (15 μM). See SI Methods for additional experimental details. (D) Representative image used to quantify in vivo replication in animals treated with either DMSO or ABT-702. Sections were stained for PDX-1 (green), BrdU (red), and DNA (blue). (E–G) Quantification of the in vivo replication rates of islet β cells (E), exocrine cells (F), and hepatocytes(G). Error bars represent the SD; P values were obtained using two-tailed t test.