Reestablishment of Glucose Inhibition of Glucagon Secretion in Small Pseudoislets

Abstract

Misregulated hormone secretion from the islet of Langerhans is central to the pathophysiology of diabetes. Although insulin plays a key role in glucose regulation, the importance of glucagon is increasingly acknowledged. However, the mechanisms that regulate glucagon secretion from α-cells are still unclear. We used pseudoislets reconstituted from dispersed islet cells to study α-cells with and without various indirect effects from other islet cells. Dispersed islet cells secrete aberrant levels of glucagon and insulin at basal and elevated glucose levels. When cultured, murine islet cells reassociate to form pseudoislets, which recover normal glucose-regulated hormone secretion, and human islet cells follow a similar pattern. We created small (∼40-µm) pseudoislets using all of the islet cells or only some of the cell types, which allowed us to characterize novel aspects of regulated hormone secretion. The recovery of regulated glucagon secretion from α-cells in small pseudoislets depends upon the combined action of paracrine factors, such as insulin and somatostatin, and juxtacrine signals between EphA4/7 on α-cells and ephrins on β-cells. Although these signals modulate different pathways, both appear to be required for proper inhibition of glucagon secretion in response to glucose. This improved understanding of the modulation of glucagon secretion can provide novel therapeutic routes for the treatment of some individuals with diabetes.

Figure 1

Formation of mouse and human pseudoislets in vitro. A: Representative images of mouse pseudoislets forming in vitro over 72 h after dispersion. Islet cells begin to aggregate within an hour of dispersion, form elongated structures at 24 h, and continue to round at 72 h. B: Islet cells, under these culture conditions, formed pseudoislets that averaged ∼40 µm at 72 h. C and D: Glucagon and insulin secretion in response to 1 and 11 mmol/L glucose was measured from mouse intact islets, dispersed (Disp.) islets, and pseudoislets at 24, 48, and 72 h (islets, dispersed cells, and pseudoislets each from six mice). E and F: Glucagon and insulin secretion in response to 1 and 11 mmol/L glucose was measured from human donor islets, dispersed islets, and pseudoislets at 3, 7, and 14 days (islets and pseudoislets from six donors) (Supplementary Table 1). Error bars represent SEM. *Differences between 1 and 11 mmol/L glucose. #Differences between control and conditions at the same glucose concentration (P < 0.05 by one-way ANOVA).

Figure 2

Changes in calcium dynamics during pseudoislet formation. A: Representative image of a Fluo-4–labeled pseudoislet. α-Cells were identified by the presence of transgenic RFP expression. B: Representative traces of Fluo-4 intensity changes in an α-cell and one β-cell within a pseudoislet at 11 mmol/L glucose. The percent of oscillating β- (C) or α- (D) cells represent data from three mice (>25 β-cells/mouse or >10 α-cells/mouse). Disp., dispersed cells. Error bars represent SEM. *Differences between 1 and 11 mmol/L glucose. #Differences between control and conditions at the same glucose concentration (P < 0.05 by one-way ANOVA).

Figure 3

Changes in cAMP levels, F-actin density, and glucagon secretion in pseudoislets. A: Fixed samples were stained for cAMP (green) and F-actin (magenta), and fluorescence intensities were measured in glucagon-positive regions (white overlay). Representative images show staining for an islet and pseudoislet. The observed changes in fixed pseudoislet morphology compared with living samples is the result of fixation. The fixed pseudoislets remain contiguous. Quantification of α-cell–specific cAMP (B) and F-actin (C) levels in intact islets, dispersed (Disp.) cells, and pseudoislets at 1 and 11 mmol/L glucose. Quantification of α-cell–specific cAMP and F-actin levels in pseudoislets at 1 and 11 mmol/L glucose in response to 100 μmol/L IMBX and 50 μmol/L Fsk, 4 µg/mL ephrinA5-Fc, or both (Dual), normalized to islet cAMP (D) or F-actin (E) levels at 1 mmol/L glucose (n = 3 mice with >15 α-cells/mouse). Glucagon secretion at 1 and 11 mmol/L glucose was measured from mouse intact islets (F) and pseudoislets at 72 h (G) in response to 100 μmol/L IMBX and 50 μmol/L Fsk, 4 µg/mL ephrinA5-Fc, or both (n = 6 mice). Error bars represent SEM. *Differences between 1 and 11 mmol/L glucose. #Differences between control and conditions at the same glucose concentration (P < 0.05 by one-way ANOVA).

Figure 4

Selective pseudoislets recapitulate aspects of the islet. A: Representative image of an α-/β-cell pseudoislet at 72 h. α-Cells (red) were identified by transgenic RFP expression, whereas β-cells (green) were identified by GFP expression. B: Glucagon secretion from α-/β-cell pseudoislets treated with 1 or 11 mmol/L glucose with or without 100 nmol/L somatostatin (SST), 12.5 μmol/L DPHBA, or 1 μmol/L S961 (n = 6 mice). C: Glucagon secretion from α-cells in coculture with δ-cells treated with 1 or 11 mmol/L glucose with or without 1 μmol/L insulin (INS), 4 µg/mL ephrinA5-Fc, or both (Dual; n = 5 mice). D: Glucagon secretion from α-cells treated with 1 or 11 mmol/L glucose with or without 1 μmol/L insulin and 100 nmol/L somatostatin, 4 µg/mL ephrinA5-Fc, or both (Dual) (n = 4 mice). E: Representative image of β-cell pseudoislets at 72 h. F: Insulin secretion from β-cell pseudoislets treated with 1 or 11 mmol/L glucose (n = 6 mice). Error bars represent SEM. *Differences between 1 and 11 mmol/L glucose. #Differences between control and conditions at the same glucose concentration (P < 0.05 by one-way ANOVA).