Fig. 2: Metabolites upstream of PEP fails to drive glucose-dependent response in SC-β cells.

A, B Insulin secretion in response to 20 mM glyceraldehyde (GLA) (A, n = 3 mice, 5 batches of SC-islets, p < 0.001 on the left graph and p = 0.037 on the right graph) or 20 mM dihydroxyacetone (DHA) (B, n = 4 mice, 4 batches of SC-islets, p = 0.0286). C–F Average [Ca²⁺]ᵢ responses to 20 mM GLA (C, n = 19 mouse β cells, 14 SC- β cells respectively from 3 different experiments, p = 0.0202), 20 mM DHA (D, n = 8 mouse β cells, 8 SC- β cells respectively from 3 different experiments, p = 0.0072), 20 mM glyceric acid (GA) (E, n = 14 mouse β cells, 18 SC- β cells respectively from 3 different experiments, p = 0.0474), and 20 mM 3-PG (F, n = 30 mouse β cells, 11 SC- β cells respectively from 3 different experiments, p = 0.0282). G Representative traces (left), and average total normalized exocytotic responses of β-cell (right) with infusion of 50 \({{\rm{\mu }}}\)M pH-corrected 3-PG, 2-PG, and K⁺-PEP from mouse-β (n = 20, 15, 15, 17 cells) and SC-β (n = 11, 12, 13, 15 cells) at 2.8 mM glucose, p = 0.04, 0.002, <0.001, <0.001. H Representative traces (left), and average Ca²⁺ currents of β-cell (right) with infusion (dialysis) of 50 µM pH-corrected 3-PG, 2-PG, and K⁺-PEP from mouse-β (n = 20, 15, 15, 17 cells) and SC-β (n = 11, 12, 13, 15 cells), p < 0.001, 0.004, <0.001, <0.001. Glucose level was 2.8 mM unless indicated. For [Ca²⁺]_i recordings, single mouse-β cell was loaded with Cal-500 AM and identified by immunostaining for insulin. Single SC-β cell was identified by using cells differentiated from H1-Ins-jGCaMP7f⁴⁶. [Ca²⁺]_i recordings were analyzed using smooth dynamics method⁵⁹. To demonstrate the functional viability of β cells and response to KCl, 5 mM KCl and 20 mM K⁺-PEP (Phosphoenol-pyruvate PEP, monopotassium salt) were added at the end of [Ca²⁺]_i recording. For patch clamp, single SC-β cell was identified by using cells differentiated from H1-Ins-EGFP. All data are presented as mean ± SEM, with statistical significance determined using unpaired two-tailed Mann–Whitney test (right graphs from A, B), unpaired two-tailed Student’s t test (right graphs from C–F), or two-way ANOVA followed by Sidak’s multiple comparison test (left graph from A, right graphs from G and H). *P < 0.05, **P < 0.01, ***P < 0.001.