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.2020 Apr 20;11(1):1896.
doi: 10.1038/s41467-020-15717-8.

Paracrine control of α-cell glucagon exocytosis is compromised in human type-2 diabetes

Affiliations

Paracrine control of α-cell glucagon exocytosis is compromised in human type-2 diabetes

Muhmmad Omar-Hmeadi et al. Nat Commun..

Abstract

Glucagon is released from pancreatic α-cells to activate pathways that raise blood glucose. Its secretion is regulated by α-cell-intrinsic glucose sensing and paracrine control through insulin and somatostatin. To understand the inadequately high glucagon levels that contribute to hyperglycemia in type-2 diabetes (T2D), we analyzed granule behavior, exocytosis and membrane excitability in α-cells of 68 non-diabetic and 21 T2D human donors. We report that exocytosis is moderately reduced in α-cells of T2D donors, without changes in voltage-dependent ion currents or granule trafficking. Dispersed α-cells have a non-physiological V-shaped dose response to glucose, with maximal exocytosis at hyperglycemia. Within intact islets, hyperglycemia instead inhibits α-cell exocytosis, but not in T2D or when paracrine inhibition by insulin or somatostatin is blocked. Surface expression of somatostatin-receptor-2 is reduced in T2D, suggesting a mechanism for the observed somatostatin resistance. Thus, elevated glucagon in human T2D may reflect α-cell insensitivity to paracrine inhibition at hyperglycemia.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1. Exocytosis of glucagon granules in normal and diabetic pancreatic α-cells.
a TIRF images of dispersed human islet cells transduced with Pppg-EGFP (top) or Pppg-NPY-EGFP (bottom). In total, 90% of EGFP expressing cells (n = 91 cells, 5 donors) and 93% of NPY-EGFP expressing cells (n = 70 cells, 4 donors) were positive for glucagon. Scale bar 2 µm.b Examples of TIRF microscopy of cells from non-diabetic (ND) and type 2 diabetic (T2D) donors expressing Pppg-EGFP together with the granule marker NPY-mCherry (gr). Examples are before and after stimulation with 75 mM K+ for 40 s. (K+ was elevated during 10–50 s). Scale bar 2 µm.c Timecourse of average cumulative number of exocytotic events normalized to cell area in experiments as inb (left); 1530 granules in 169 ND cells (black), 441 granules in 75 T2D cells (red). Exocytosis (right) was 0.055 ± 0.005 gr µm−2 in 12 T2D donors compared with 0.077 ± 0.005 gr µm−2 in 29 ND donors (p = 0.001, two-tailedt-test). Inc,d, timecourse (left) shows mean ± SEM of all cells, bargraphs (right) show donor means (dots) and mean ± SEM of individual donor means (bars).d Time courses of granule (gr) density (left) in ND or T2D cells as inb. Glucagon density (right) was 0.56 ± 0.017 gr µm−2 in 17 T2D donors compared with 0.61±0.01 gr µm−2 in 50 ND donors (p = 0.028, two-tailedt-test).e Total exocytosis during K+-stimulation plotted as function of granule density. Each symbol ine–g represents represent individual donors ± SEM (averages forn = 29 ND donors in black, andn = 12 T2D donors in red; 5–20 cells for each donor). Correlation was quantified as Pearson coefficientr (see main text).f Total exocytosis during K+-stimulation plotted as function of donor HbA1c.n = 26 ND donors andn = 10 T2D donors.g Granule density as function of donor HbA1c;n = 40 ND andn = 15 T2D donors.
Fig. 2
Fig. 2. Voltage-dependent currents and exocytosis in human α-cells.
a Families of voltage-clamp current responses in human ND and T2D α-cells. Currents were elicited by 50 ms depolarizing pulses (−70 to +80 mV in 10 mV increments) from a holding potential of −70 mV. For clarity, only the responses between −40 mV and +10 are shown.b,c Current (I)–voltage (V) relationships for Ca2+ (B, average current during 5–45 ms of the depolarization ina) and Na+ (c, peak current during the first 5 ms of the depolarization ina) currents recorded from ND (n = 38, 4 donors, black) and diabetic T2D (n = 32, 3 donors, red) cells as ina. Currents are normalized to cell size (pF). Data are presented as mean values ± SEM.d Cell capacitance increase (ΔCm) during a train of 14 × 200-ms depolarizations from −70 mV to 0 mV in ND (black) and T2D (red) α-cells.e Average change in membrane capacitance, normalized to initial cell capacitance (ΔC/C0), during the 1st depolarization (#1), and total increase during the train (Σ1-14) for ND (n = 20, 4 donors, black) and T2D (n = 18, 3 donors, red) α-cells. Data are presented as mean values ± SEM.f Whole-cell membrane capacitance (CM) in T2D (n = 48, 7 donors) and ND (n = 66, black, 8 donors) α-cells. Dots represent individual cells, and lines are mean values. Each donor is represented by a single color.
Fig. 3
Fig. 3. Glucose dependence of α-cell exocytosis.
a Examples TIRF images of dispersed ND (left) and T2D α-cells (right) expressing Pppg-NPY-EGFP, after equilibration in the indicated glucose concentrations. Scale bar 2 µm.b Average exocytosis as function of ambient glucose concentration for dispersed ND (black) and T2D (red) α-cells as ina. For ND,n = 13 cells/3 donors at 1 mM, 8 cells/2 donors at 3 mM, 13 cells/2 donors at 7 mM, 15 cells/3 donors at 10 mM, and 10 cells/2 donors at 20 mM). For T2D,n = 7 cells/2 donors at 1 mM), 9 cells/2 donors at 3 mM, 10 cells/2 donors at 7 mM, 10 cells/2 donors at 10 mM, and 7 cells/2 donors at 20 mM. Data are presented as mean values ± SEM.c Docked granules (average granule density) as function of ambient glucose concentration for ND (black,n = 32 cells/5 donors at 1 mM, 32 cells/5 donors at 3 mM, 32 cells/5 donors at 7 mM, 32 cells/5 donors at 10 mM, and 32 cells/5 donors at 20 mM) and for T2D cells (red,n = 11 cells/2 donors, 11 cells/2 donors at 3 mM, 17 cells/2 donors at 7 mM, 16 cells/3 donors at 10 mM, and 11 cells/2 donors at 20 mM). Data are presented as mean values ± SEM. Inb,cp-values in black or red for comparisons as indicated by a line in ND group or T2D group respectively (two-tailedt-test).d Average rate of docking (granules becoming immobilized in the TIRF plane) as function of the ambient glucose concentration in the same cells as inb. Data are presented as mean values ± SEM.e Cumulative time course (upper), total exocytosis (middle), and initial density of docked granules (lower) during K+-stimulated exocytosis in dispersed ND α-cells bathed in 1 mM (30 cells/6 donors, yellow), 7 mM (38 cells/6 donors, blue) or 10 mM glucose (71 cells/14 donors, black). Stimulation was from 10 to 50 s. Data are presented as mean values ± SEM.f As ine, but for dispersed T2D α-cells.n = 27 cells/5 donors at 1 mM, 24 cells/3 donors in 7 mM, and 33 cells/6 donors in 10 mM glucose. Ine,f,p-values in black for comparisons as indicated by a line, or in red comparing ND and T2D for the same condition (oneway ANOVA, Fisher posthoc test).
Fig. 4
Fig. 4. Disturbed paracrine signaling in α-cells within intact islets.
a TIRF images of NPY-EGFP of an α-cell within an intact islets of ND (left, representative for 67 cells) or T2D (right, representative for 66 cells) human donors. Scale bar 2 µm. Lower: image sequence of an exocytosis event in the ND α-cell example.b Average spontaneous exocytosis in α-cells within intact ND islets that were bathed in 1 mM (11 cells/3 donors, black), 7 mM (10 cells/3 donors, black), or 10 mM glucose (16 cells/4 donors, black), and in 10 mM glucose with SSTR antagonist (200 nM CYN154806, 19 cells/3 donors, light blue) or insulin receptor antagonist (1 μM S961, 11 cells/2 donors, green). Data are presented as mean values ± SEM. Scale bar 1 µm.c As inb, but for α-cells within intact T2D islets at 1 mM (21 cells/5 donors), 7 mM (16 cells/4 donors), and 10 mM glucose (29 cells/5 donors). Inb,c,p-values are indicated for selected comparisons (one-way ANOVA with Fisher posthoc test). Data are presented as mean values ± SEM.d Representative confocal images of human pancreatic tissue sections of ND donors (top) and T2D donors (bottom), co-immunostained anti-SSTR2 (green) and anti-glucagon (red); scale bar 10 µm. The white square indicates the area that is enlarged in the right-most images (SSTR2; scale bar 2 µm).e Average SSTR2 staining intensity (F-background, average pixel value pxl), measured along a line across the plasma membrane of 828 cells from 5 ND donors (black) and 824 cells from 5 T2D donors (red). Cells were spatially aligned so that the line crosses the center-right of the cell perimeter at distance zero (illustrated drawing, top). Dots indicate average donor values. Staining intensities at distance zero were significantly different between ND and T2d donors (p = 4 × 10−11, two-tailedt test).f As inb–e, but for glucagon staining.
Fig. 5
Fig. 5. Paracrine regulation of exocytosis in dispersed α-cells.
a Cumulative time course (upper), total exocytosis (middle), and initial density of docked granules (lower) during K+-stimulated (gray bar) exocytosis in dispersed ND α-cells in control conditions (black, 10 mM glucose,n = 71 cells/14 donors) or exposed to somatostatin (light blue, SST, 400 nM,n = 53 cells/9 donors), insulin (green, INS, 100 nM,n = 53 cells/8 donors), forskolin (purple, FSK, 2 µM,n = 30 cells/5 donors), GABA (brown, 400 nM,n = 14 cells/3 donors), adrenaline (pink, ADR, 5 µM,n = 30 cells/5 donors), glutamate (orange, Glut, 1 mM,n = 16 cells/3 donors). Ina–d, significant differences compared with control are indicated withp-values (one-way ANOVA, Fisher posthoc test). Data are presented as mean values ±SEM.b As in A, but for dispersed T2D α-cells. T2D ctrln = 33 cells/6 donors, T2D SSTn = 26 cells/5 donors, T2D INSn = 19 cells/4 donors, T2D ADRn = 19 cells/3 donors.c,d As ina,b, but in presence of 1 mM glucose. ND ctrln = 30 cells/6 donors, ND SSTn = 27 cells/6 donors, ND INSn = 24 cells/5 donors, ND ADRn = 15 cells/3 donors, ND Glutn = 9 cells/2 donors, T2D ctrln = 27 cells/5 donors, T2D SSTn = 17 cells/3 donors, T2D INSn = 23 cells/3 donors, and T2D ADRn = 10 cells/2 donors.
Fig. 6
Fig. 6. Time course of paracrine inhibition in dispersed α-cells.
a Time course of spontaneous exocytosis for a representative ND α-cell bathed in 10 mM glucose (black) and challenged with somatostatin during the indicated interval (SST, 400 nM; left, blue bar). Bars to the right show quantification of average exocytosis during the three time periods of the experiment (36 cells/6 donors). Data are presented as mean values ±SEM. Ina–d, significant differences are indicated withp-values (one-way ANOVA, Fisher posthoc test).b As ina, but for T2D α-cells (38 cells/6 donors).c,d As inb–e, but challenged with insulin (INS, 100 nM, green bar). 21 cells/4 ND donors and 25 cells/3 T2D donors.e,f Example (upper) and average (lower) membrane potential recording in dispersed ND α-cells bathed in 10 mM glucose. Somatostatin (SST, 400 nM, 17 cells/4 donors, blue shading ine) or insulin (INS, 100 nM, 25 cells/6 donors, green shading inf) were applied during the indicated time interval.g,h as ine,f, but for dispersed T2D α-cells (11 cells/2 donors in G; 13 cells/4 donors inh).
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