Volume 27, Issue 2, Pages 470-478.e4 (February 2018) Glucose-Dependent Granule Docking Limits Insulin Secretion and Is Decreased in Human Type 2 Diabetes  Nikhil R. Gandasi, Peng Yin, Muhmmad Omar-Hmeadi, Emilia Ottosson Laakso, Petter Vikman, Sebastian Barg  Cell Metabolism  Volume 27, Issue 2, Pages 470-478.e4 (February 2018) DOI: 10.1016/j.cmet.2017.12.017 Copyright © 2018 Elsevier Inc. Terms and Conditions

Cell Metabolism 2018 27, 470-478.e4DOI: (10.1016/j.cmet.2017.12.017) Copyright © 2018 Elsevier Inc. Terms and Conditions

Figure 1 Docked Granules Limit Insulin Secretion and Are Reduced in T2D (A) Cells from non-diabetic (ND) or type 2 diabetic (T2D) donors expressing NPY-mCherry, imaged by TIRF microscopy before and after stimulation with either 10 mM glucose for 20 min or 75 mM K+ for 40 s. Scale bar, 1 μm. (B) Image sequences (0.1 s per frame, 1.25 μm2) showing two individual granules in the glucose-stimulated cell (ND in A) that either underwent exocytosis (exo, top) or failed to do so (fail, bottom). (C and D) Time courses of exocytosis frequency (top, rolling average of 6 s in C and 2 s in D), cumulative exocytosis (middle), and granule density (lower) in ND or T2D cells as in (A). Cells were stimulated with 10 mM glucose (C) or 75 mM K+ (D) from t = 0. Shaded areas represent 1 SEM. (E) Cumulative exocytosis at the end of the 40 s K+ stimulation in (D). (F) Average granule density in unstimulated ND or T2D cells. In (E–I), symbols represent individual donors ± SEM (5–50 cells for each donor), bars represent group means, and shaded areas the group SEMs; ∗∗∗ indicates p<0.001 (t test). (G–I) Granule density as a function of exocytosis (G, K+ stimulation), islet insulin stimulation index (SI) (H), or donor HbA1c (I). ND donors are shown in black, T2D in red. Blue lines represent linear fits to the entire dataset. Cell Metabolism 2018 27, 470-478.e4DOI: (10.1016/j.cmet.2017.12.017) Copyright © 2018 Elsevier Inc. Terms and Conditions

Figure 2 Slow Recovery of Docked Granules after Exocytosis (A) TIRF images of two ND cells expressing NPY-mCherry challenged with a dual-pulse protocol (2 × 75 mM K+ for 40 s, as shown in C). The upper cell rested 2 min, and the lower 20 min, before the second stimulation. (B) Cumulative exocytosis for first (dark) and second stimulation (light), with 2 min (left, 17 cells) or 20 min (right, 8 cells) resting intervals, as in (A). Shaded areas indicate SEM. (C) Quantitative analysis of (A) and (B). Stimulation protocols (top), granule density (circles), and cumulative exocytosis during the two stimulations (bars, “exo”); data for 2 min (dark) and 20 min (light) intervals are shown. Error bars indicate SEM. (D–F) As in (A)–(C), but for T2D cells. Scale bar in (D), 1 μm; (F) ∗∗∗p < 0.001 versus ND. Cell Metabolism 2018 27, 470-478.e4DOI: (10.1016/j.cmet.2017.12.017) Copyright © 2018 Elsevier Inc. Terms and Conditions

Figure 3 Glucose-Dependent Modulation of Docking Is Absent in T2D (A) Density of docked granules in ND cells exposed to the conditions specified (3G or 10G, 3 or 10 mM glucose; Fsk, forskolin; SST, somatostatin; CCh, carbachol; PMA, phorbol ester; Dz, diazoxide). ∗∗∗p < 0.001 for 3 versus 10 mM glucose with the same drug. Error bars indicate SEM. (B) Time course of docked granule density after switching from 3 to 10 mM glucose, in presence of diazoxide. Each point corresponds to a single ND cell, from 13 coverslips. Purple line is a mono-exponential fit with τ = 9.6 ± 3.8 min. (C) Time course of cumulative K+-stimulated exocytosis (10 mM glucose, diazoxide) in presence (blue) or absence (purple) of 10 nM exendin-4. Shaded areas indicate SEM. (D) Average exocytosis frequency (0–40 s) in experiments as in (C), for the indicated conditions. ∗∗∗p < 0.001. (E) Primed granules as fraction of docked granules, obtained by dividing exocytosis values as in (C) with docked granule density as in (A). ∗∗∗p < 0.001 (t test). Error bars in (D and E) indicate SEM. (F–J) As in (A)–(E), but for T2D cells. (J) ∗p < 0.05. ∗∗∗p < 0.001 versus the same condition in ND cells or as indicated. (K) Image sequences (1 s per frame, 1.25 μm2) showing single granules during docking, visiting, and undocking in an ND cell exposed to 10 mM glucose (with diazoxide). (L) Granule residence times at the plasma membrane (>2 s), shown as survival plots for ND cells in 3 mM (blue) or 10 mM glucose (red) cells. Events starting during the final 150 s of each movie were ignored. Shaded areas indicate SEM. (M) Frequencies (±SEM) of successful docking events (residence >90 s) in (L), normalized for observation time. The value for glucose-stimulated exocytosis (Figure 1E) is shown for comparison. ∗∗∗p < 0.001. (N–P) as in (L) and (M), but for T2D cells. ∗p < 0.05 for indicated comparison; ∗∗∗p < 0.001 versus ND. (Q) Undocking frequency as function of docking frequency in stimulatory (white: 10 mM glucose) and non-stimulatory conditions (blue, 10 mM glucose + Dz; black, 3 mM glucose). (R) Survival function (red, Kaplan-Meier estimator with interval censoring) and 95% confidence intervals (dotted) for priming of 102 newly docked granules (10 mM glucose, 2 μM Fsk, 0.2 mM Dz). ND cells were observed for 6–12 min followed by stimulation with 75 mM K+ for 10 s. All granules that docked during the observation period were analyzed and scored as primed when they exocytosed during the stimulation. Cell Metabolism 2018 27, 470-478.e4DOI: (10.1016/j.cmet.2017.12.017) Copyright © 2018 Elsevier Inc. Terms and Conditions

Figure 4 Decreased Gene Expression Related to Docking in T2D Donors (A and B) Pearson correlation coefficients (Corr co-eff) of mRNA counts for the indicated genes (quantified by RNA-seq) and donor HbA1c (A) or insulin stimulation index (SI) (B). At least three donors with ≥20 cells each were used. (C) Pearson correlation coefficients of docked granule density versus expression of exogenous EGFP-tagged proteins. Expression was quantified as background-corrected cellular EGFP fluorescence. In (A)–(C), color indicates p value of the correlations; asterisks mark significance after Holm-Bonferroni correction. Cell Metabolism 2018 27, 470-478.e4DOI: (10.1016/j.cmet.2017.12.017) Copyright © 2018 Elsevier Inc. Terms and Conditions