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. 2012 Mar 6;109(10):3915-20.
doi: 10.1073/pnas.1201149109. Epub 2012 Feb 15.

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

Justin P Annes  1 , Jennifer Hyoje Ryu, Kelvin Lam, Peter J Carolan, Katrina Utz, Jennifer Hollister-Lock, Anthony C Arvanites, Lee L Rubin, Gordon Weir, Douglas A Melton
Affiliations

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

Justin P Annes et al. Proc Natl Acad Sci U S A. .

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.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
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.
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.
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.
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.
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.
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