Kalirin/Trio Rho GDP/GTP exchange factors regulate proinsulin and insulin secretion

in Journal of Molecular Endocrinology
Correspondence should be addressed to R Kuliawat: regina.kuliawat@einstein.yu.edu
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Key features for progression to pancreatic β-cell failure and disease are loss of glucose responsiveness and an increased ratio of secreted proinsulin to insulin. Proinsulin and insulin are stored in secretory granules (SGs) and the fine-tuning of hormone output requires signal-mediated recruitment of select SG populations according to intracellular location and age. The GTPase Rac1 coordinates multiple signaling pathways that specify SG release, and Rac1 activity is controlled in part by GDP/GTP exchange factors (GEFs). To explore the function of two large multidomain GEFs, Kalirin and Trio in β-cells, we manipulated their Rac1-specific GEF1 domain activity by using small-molecule inhibitors and by genetically ablating Kalirin. We examined age-related SG behavior employing radiolabeling protocols. Loss of Kalirin/Trio function attenuated radioactive proinsulin release by reducing constitutive-like secretion and exocytosis of 2-h-old granules. At later chase times or at steady state, Kalirin/Trio manipulations decreased glucose-stimulated insulin output. Finally, use of a Rac1 FRET biosensor with cultured β-cell lines demonstrated that Kalirin/Trio GEF1 activity was required for normal rearrangement of Rac1 to the plasma membrane in response to glucose. Rac1 activation can be evoked by both glucose metabolism and signaling through the incretin glucagon-like peptide 1 (GLP-1) receptor. GLP-1 addition restored Rac1 localization/activity and insulin secretion in the absence of Kalirin, thereby assigning Kalirin’s participation to stimulatory glucose signaling.


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    β-Cells express Kalirin and Trio, two homologous GEFs implicated in secretory granule (SG) maturation and exocytosis. (A) Illustration of the multidomain structure shared by Kalirin and Trio: Sec14, Sec14 homology domain; DH, Dbl homology domain; PH, pleckstrin homology domain; SH3, SRC homology 3 domain, Ig, immunoglobulin domain; FN, fibronectin domain. Major Kalirin and Trio splice variants are shown. Regions recognized by the Kalirin (Sec14) and Trio antibodies (spectrin repeats 5 and 6) or targeted by primer pairs (red arrows) in quantitative PCR (qPCR) reactions are indicated. (B) The relative expression of Kalirin and Trio isoforms in mouse islets, β-cell lines and pituitary as determined by qPCR and normalized to Gapdh. (C) (i) Antiserum to the N-terminus of Kalirin (brackets in A) determines Kalirin distribution within mouse pancreatic islets. Phalloidin stained cortical actin was used to outline cells (red). Insulin positive cells are identified by asterisks and arrows indicate Kalirin positive cells devoid of insulin labeling. (D) Antisera to the N-termini of Kalirin and Trio (brackets in A) determine their cellular localization in βTC3 cells (Kalirin, green; Trio, red). Cells were counter stained with DAPI to visualize nuclei. Arrow indicates Kalirin labeling at cell periphery. (E) Expression of endogenous Kalirin and Trio in β-cell lines was determined by immunoblot. Cell lysates from duplicate wells were normalized to protein concentrations and Gapdh staining served as a loading control.

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    SG characterization and secretion profiling of human islets demonstrate high ISG content and the presence of ISG-derived, constitutive like vesicular pathway. (A) In the merged image, insulin is stained red, proinsulin green. (B) Electron micrographs confirm the presence of ISGs (white arrows, electron lucent content) and mature β-granules (white filled arrows, electron-dense core surrounded by halo) in human and mouse β-cells. (C) Quantitative evaluation of ISG numbers per β-cell area in human or rodent islets or INS-1 β-cell line reveals statistically significant differences in ISG content (n = 15 cells). Human vs mouse β-cells, P = 0.0003; mouse β-cells vs INS-1 cell line, P = 0.0004. (D) Radiolabeled human islets were chased in 5 mM glucose for the indicated times and immunoprecipitated peptide profiles were determined. Arrowheads indicate intracellular content of proinsulin, conversion intermediates (CI) and insulin. (E) Media content of hormone collected during three sequential 1-h intervals. (F) Pulse chase experiment repeated with islets from a different donor (media, 0–1 h). Subsequent addition of stimulatory glucose (media, 1–2 h stim). (G) After low glucose (5 mM), exocytosis (16 mM glucose) was examined (media, G: 2–3 h stim, or F: 1–2 h stim).

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    In human islets, hormone release via constitutive-like vesicular traffic or direct granule exocytosis depends on Kalirin/Trio GEF1 domain activity. (A) Radiolabeled human islets were treated or not with the small molecule inhibitor NPPD, then chased; DMSO was the solvent control. Tricine-SDS-PAGE separation of immunoprecipitated insulin-containing peptides shows hormone content of media collected during four sequential 1 h intervals (3 × 5 mM glucose, 1 × 16 mM glucose: stim) or retained in the cell (cell lys). (B) For increased sensitivity of detection, a radioautograph was obtained after long (14 day) exposure. (C) Quantification of CI, Proins and Ins from fluorograms as percent total radioactivity; stars mark significantly altered secretion. (D) Exclusion of fluorogenic peptide (bis-AAF-R110) was used to test cell membrane integrity of healthy cells and fluorescence intensity (Ex/Em: 485 nm/530 nm) was normalized to sample protein content. No differences in fluorescence signal were observed with or without NPPD (human islets and βTC3 cells). Cell permeabilization with saponin served as a positive control. A full color version of this figure is available at https://doi.org/10.1530/JME-18-0048.

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    Kalirin elimination reproduces the pharmacological GEF1 phenotype of impaired glucose-stimulated insulin secretion with an even stronger impact on ISG exocytosis. (A) For in vivo secretion studies, plasma insulin content prior to or 60 min after an intraperitoneal glucose administration was analyzed using ELISA. (B) Radiolabeled islets (30 min pulse) obtained from WT and Kal-KO mice were chased for three sequential incubations (2 × 5 mM glucose, 1–4 h and 4–5 h, followed by 1 × 16 mM glucose, 5–6 h stim). (C) Quantification of total amount of radioactive insulin recovered with stimulation. (D) To focus on the glucose response of newly synthesized granules, the chase time was shortened to 2 h. (E) Quantification. Release of newly synthesized molecules is reduced with loss of Kalirin. (F) Peptides released into media at 2 or 6 h of chase were quantitated and set up as a ratio (WT/Kal-KO). Results of three secretion experiments done in duplicate were averaged, P = 0.0408. A full color version of this figure is available at https://doi.org/10.1530/JME-18-0048.

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    Change of ISG size during maturation is Kalirin independent. (A, B and C) Electron micrographs of β-cells from WT (A) and Kal-KO mice (B and C). (A) Electron micrographs of WT or (B) Kal-KO β-cells obtained from the intact fixed pancreatic tissue reveal no differences in SG morphology. (n = 2 animals, tissue from 5 month old female mouse shown). (C) Viewed at higher magnification with particular focus on ISGs in vicinity of the Golgi complex (indicated by arrowheads and G). (D) Quantitative evaluation of beta-cell granule maturation by ultrastructural analyses.

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    RRP exocytosis bypasses the need for Kalirin. (A) Release of the RRP of granules was tested in vivo. WT and Kal-KO mice were fasted for 6 h and plasma samples were taken prior to (basal) and after a 3 min arginine challenge. No significant differences in basal insulin levels or insulin release were observed between WT and Kal-KO mice in response to arginine administration (n = 7 animals). (B) Islets isolated from WT and Kal-KO mice were exposed for 30 min to 5 mM glucose or stimulatory (11 mM glucose plus 30 mM KCl) conditions. Basal insulin secretion from Kal-KO islets was reduced (WT: 0.24 ± 0.01, vs Kal-KO: 0.12 ± 0.02, P = 0.0002). Kal-KO and WT islets were equally capable of secreting insulin in response to glucose plus KCl (WT: 3.2 ± 0.70, Kal-KO: 3.5 ± 0.46, P = 0.5534).

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    Drugs targeting Kalirin/Trio GEF1 domain alter Rac1 localization and activity. Simultaneous rescue of Rac1 function and insulin output requires GLP-1 signaling. (A) Rac1 distribution in βTC3 cells (5 mM G, panels ii, and iii; 10 min at 16 mM G, panels ii and iii) relative to the plasma membrane marker NKA (panel i) was determined by immunofluorescence labeling. (B) NKA:Rac1 colocalization at the cell periphery was quantified using CellProfiler and Pearson’s correlation coefficients (PCC) as described in ‘Methods’ section. Data (n ≥ 100 cells per condition) are mean ± s.e.m. PCC values increased with glucose stimulation (16 mM G: 0.84 ± 0.03 vs 5 mM G: 0.22 ± 0.06, P < 0.0001) and were attenuated with ITX3 pretreatment (16 mM G + ITX3: 0.34 + 0.07 vs 5 mM G: 0.22 + 0.06, P = 0.24; vs 16 mM G alone: 0.84 + 0.03, P < 0.0001). No significant differences were observed when GLP-1 plus high glucose were used as stimulus (16 mM G + GLP-1, no ITX3: 0.71 + 0.06, vs 16 mM G + GLP-1, plus ITX3: 0.75 + 0.05, P = .72). (C) Glucose response of a Rac1 FRET reporter. Small squares denote areas that were enlarged and shown as a magnified area in the lower panel. Redistribution of the reporter ± ITX and ± GLP-1 is shown. (D) FRET/donor intensities of the Rac1 FRET reporter within 2.2 µm from the cell’s edge were measured and set up as a ratio to the average FRET/donor intensity in the rest of cell (n = 14/condition). Control vs ITX: P = 0.0027; 16 mM G + GLP-1, P = 0.6663. (E) Secretion studies using static incubations of WT or Kal-KO islets exposed to 5 mM glucose or for 10 min to stimulatory (16 mM glucose or 16 mM glucose plus 10 µM GLP-1) conditions. Control vs Kal-KO: P = 0.0489; 16 mM G + GLP-1, P = 0.0730.


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