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Rab26 restricts insulin secretion via sequestering Synaptotagmin-1 [1]

['Ruijuan Zhuang', 'School Of Pharmaceutical Sciences', 'State Key Laboratory Of Cellular Stress Biology', 'Fujian Provincial Key Laboratory Of Innovative Drug Target Research', 'Xiamen University', 'Fujian', 'Yuxia Zhou', 'School Of Basic Medical Sciences', 'Guizhou Provincial Key Laboratory Of Pathogenesis', 'Drug Research On Common Chronic Diseases']

Date: 2023-06

Rab26 is known to regulate multiple membrane trafficking events, but its role in insulin secretion in pancreatic β cells remains unclear despite it was first identified in the pancreas. In this study, we generated Rab26 -/- mice through CRISPR/Cas9 technique. Surprisingly, insulin levels in the blood of the Rab26 -/- mice do not decrease upon glucose stimulation but conversely increase. Deficiency of Rab26 promotes insulin secretion, which was independently verified by Rab26 knockdown in pancreatic insulinoma cells. Conversely, overexpression of Rab26 suppresses insulin secretion in both insulinoma cell lines and isolated mouse islets. Islets overexpressing Rab26, upon transplantation, also failed to restore glucose homeostasis in type 1 diabetic mice. Immunofluorescence microscopy revealed that overexpression of Rab26 results in clustering of insulin granules. GST-pulldown experiments reveal that Rab26 interacts with synaptotagmin-1 (Syt1) through directly binding to its C2A domain, which interfering with the interaction between Syt1 and SNAP25, and consequently inhibiting the exocytosis of newcomer insulin granules revealed by TIRF microscopy. Our results suggest that Rab26 serves as a negative regulator of insulin secretion, via suppressing insulin granule fusion with plasma membrane through sequestering Syt1.

Funding: This work was supported by the National Natural Science Foundation of China (grants 31871423 and 91954111 to WT), the National Natural Science Foundation of China (grants 82000741 and 32160207 to ZY), and the National Natural Science Foundation of China (grants 31871386 and 32070736 to CX). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Copyright: © 2023 Zhuang et al. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

In this study employing Rab26 gene knockout mice, we found that deficiency of Rab26 enhances insulin secretion. Conversely, overexpression of Rab26 inhibits insulin secretion in pancreatic insulinoma cells and freshly isolated mouse islets. Rab26 overexpression induces clustering of insulin granules. Mechanistically, Rab26 interacts with Synaptotagmin-1 (Syt1), and this interaction may competitively inhibit Syt1 binding to the SNARE complex to interfere insulin granule fusion to plasma membrane.

Several studies revealed that Rab26 plays important roles in regulating protein transport. Rab26 is involved in autophagic pathway by regulating autophagic degradation of phosphorylated Src and also linking synaptic vesicles to the autophagy pathway [ 13 , 14 ]. Rab26 modulates the transport of α2-adrenergic receptors from the Golgi to the cell surface [ 15 ]. Rab26 may also regulate maturation of exocrine granules and may be involved in the recruitment of secretory granules to the plasma membrane to mediate amylase release in rat parotid acinar cells [ 16 ]. These studies suggest a potential role of Rab26 in regulating other secretory events such as insulin secretion.

Rab small GTPases associate with different subcellular compartments of the exocytotic and endocytic pathways [ 7 ]. Rab proteins are the key regulators for vesicular trafficking via serving as the molecular switches cycling between GDP-bound and GTP-bound form [ 8 , 9 ]. GTP-bound form generally engages downstream effectors to regulate vesicle formation and/or tethering or docking. The biogenesis of insulin granules (dense core granules) and insulin secretion are regulated by Rab proteins and their effectors. Two Rab proteins, Rab3 and Rab27, are associated with insulin granules, which interact with multiple effectors to regulate insulin secretion through mediating granule trafficking, docking to the plasma membrane [ 10 ]. Especially, mutation in Rab27 caused diabetes in mice [ 11 ]. Rab26 was firstly characterized from rat pancreas [ 12 ]; however, its role in pancreatic β cells remain unclear.

Insulin is the most important hormone to maintain glucose homeostasis. Defect in insulin secretion is the main pathophysiological mechanism accounting for diabetes mellitus [ 1 ]. Islet β cells, a small number of endocrine cells comprising less than 2% of the human pancreas in adults [ 2 ], secrete insulin in a biphasic manner in response to secretagogues [ 3 ]. Insulin secretion includes a series of vesicular trafficking processes tightly regulated by multiple membrane trafficking machineries such as Rab small GTPases, SNARE proteins, and SNARE associated partners. Reduced insulin secretion could be due to either defects in insulin granule transport (granule maturation), docking to or fusion with the plasma membrane, or abnormal degradation of insulin/proinsulin [ 3 – 5 ]. Dysfunction in mitochondria with loss of ATP also results in the impaired glucose-stimulated insulin secretion [ 6 ].

Results

Deficiency of Rab26 in mice improves glucose homeostasis Insulin is the most important hormone for regulating glucose homeostasis; therefore, we examined the effects of depletion of Rab26 on glucose tolerance in mice. We first performed intraperitoneal glucose tolerance test (IPGTT) to assess the consequence of Rab26 depletion. As shown in Fig 1I and 1J, Rab26-/- mice have higher insulin levels in the blood upon glucose stimulation than wild-type (WT) mice, and the glucose level in the plasma of Rab26-/- mice is much lower than that of WT mice under normal feeding condition (Fig 1K and 1L). The body weight of Rab26-/- mice is less than that of WT mice (S1G Fig). Intraperitoneal insulin tolerance tests (IPITTs) demonstrated that Rab26-/- is more sensitive to insulin (Fig 1M and 1N), since the blood glucose levels lowered down faster in Rab26-/- mice. Together with the above results, deficiency of Rab26 may improve glucose homeostasis to prevent diabetes development through enhanced insulin secretion.

Rab26 associates with insulin granules and regulates the distribution of insulin granules To investigate how Rab26 regulates insulin secretion, we examined the subcellular location of Rab26 and the effects of its overexpression on the distribution of insulin granules in insulinoma cells. Rab26 was shown to be associated with the endocytic compartments [18]. Immunostaining results for the endogenous Rab26 using Rab26 antibody revealed that over 50% Rab26-containing vesicles colocalize with insulin granules under different glucose conditions in INS-1 cells (Fig 4A). In addition, immunofluorescence microscopy revealed that GFP-Rab26 was present in puncta, which colocalized with pro (insulin) and Vamp4 in MIN6 cells (Fig 4B). These results suggest that Rab26 physiologically associates with the secretory vesicles and insulin granules and is supposed to be a regulator for insulin secretion. Further examinations demonstrated that overexpression of Rab26 resulted in insulin granules clustering in normal media, KRB buffer containing 2.8 mM glucose or 16.7 mM glucose in MIN6 cells (Fig 4C). Under normal conditions, overexpression of Rab26WT or Rab26Q123L mutant induced insulin granules clustering, while the inactivated mutant Rab26T77N was distributed in the cytosol and did not induce insulin granule granules clustering (Fig 4D). Consistent with the results that Rab26WT and Rab26Q123L but not Rab26T77N inhibit insulin secretion, this clustering distribution of insulin granules induced by Rab26 may inhibit glucose stimulation triggered exocytosis of insulin granules and consequently restrict insulin secretion. PPT PowerPoint slide

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TIFF original image Download: Fig 4. Rab26 induces clustering and enlargement of insulin particles. (A) INS-1 cell were double-labeled with anti-Rab26 and anti-insulin under 2.8 mM, 16.7 mM, or 33.4 mM glucose condition. (B) MIN6 cells transfected with GFP-Rab26 were cultured in KRB buffer containing 16.7 mM glucose for 40 min, and immunofluorescence microscopy revealed that GFP-Rab26 was expressed in puncta, which colocalized with pro(insulin) and Vamp4. (C) In MIN6 cells transiently transfected plasmids, insulin were similarly dispersed throughout the cytosol and displayed a similar extent of colocalization with Rab26WT. (D) MIN6 cells were transfected with GFP-Rab26WT, Rab26T77N, or Rab26Q123L and labeled with insulin. Bar = 20 μm. https://doi.org/10.1371/journal.pbio.3002142.g004

Rab26 interacts with synaptotagmin-1 To uncover the molecular mechanism for Rab26 in regulating insulin secretion, we searched for its interacting proteins. Syt1 along with several other proteins were identified as potential interacting partners of Rab26 from rat brain tissue lysate in large scale GST-pulldown experiments (S3A Fig). Syt1 is an important calcium sensor to regulate neurotransmitter release in neuron and insulin secretion in pancreatic β-cells [19,20]. To confirm the interaction between Rab26 and Syt1, we performed co-IP experiments using Syt1 antibody to precipitate the endogenous Rab26 in INS-1 cell lysates (Fig 5A); the results revealed that Rab26 interacts with Syt1 in INS-1 cells. The interaction of Rab26 with Syt1 was validated by analytic GST-pulldown assay. The interaction between Rab26 and Syt1 depends on Rab26’s nucleotide binding activity, as the interaction between GDP-bound mutant Rab26T77N and Syt1 was dramatically reduced (Fig 5B). In vitro binding assay using prokaryotic-expressed GST-Syt1 and His-Rab26 showed that Rab26 can directly bind to Syt1 (Fig 5C). In addition, GST-pulldown experiments using GST-Rab26 revealed that Rab26 only interacts with Syt1-C2A domain, but not C2B domain in cell lysates derived from cells transfected with GFP-Syt1, GFP-Syt1-C2A, or GFP-Syt1-C2B (Fig 5D). Again, Rab26 binds directly Syt1 through interacting with the C2A domain in a manner dependent on its binding with GTP (Fig 5B). Besides Syt1, other members of Synaptotagmin such as Syt4 and Syt7 may regulate insulin secretion as well [21–23]. However, Rab26 did not interact with Syt4 or Syt7 (S3B and S3C Fig), suggesting that Rab26 specifically interacts with Syt1. PPT PowerPoint slide

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TIFF original image Download: Fig 5. Rab26 directly interacts with Syt1. (A) co-IP experiments using Syt1 antibody to precipitate the endogenous Rab26 in INS-1 cell lysates. (B) GST, GST-Rab26WT, GST-Rab26T77N, and GST-Rab26Q123L were mixed with the 293T cell lysates containing GFP-Syt1, Syt1-C2A, or C2B, respectively. Western blotting analysis was performed with GFP antibody. (C) In vitro binding assay showed His-Rab26 can directly bind to GST-Syt1. (D) GFP-Syt1 and GST-Vector, GFP-Syt1 and GST-Rab26, GFP-Syt1-C2A and GST-Rab26, GFP-Syt1-C2B and GST-Rab26, cell lysate was mixed, and after extensive washing. After a large number of washing, western blotting analysis was performed with GFP antibody. (E) MIN6 cells transfected with GFP-Rab26 were cultured in normal medium or in KRB buffer containing 16.7 mM glucose for 40 min and then immunostained with antibody against Syt1 and insulin, showing that Rab26 induces Syt1 and insulin granules clustering. Bar = 20 μm. https://doi.org/10.1371/journal.pbio.3002142.g005

Rab26 influences the interaction between Syt1 and SNARE complex Since Syt1 facilitates secretory vesicles docking to and fusion with plasma membrane through interaction with SNARE complex [25], we next examined whether Rab26 influences the interaction between Syt1 and SNARE complex. 293t cells were cotransfected with myc-Rab26 and GFP-SNAP25 or GFP-Syntaxin-1; the cell lysates were subjected for GST-pulldown using GST-Syt1. As shown in Fig 7A and 7B, the amount of SNAP25 protein bound to GST-Syt1 was significantly reduced in cells expressing Rab26 compared with control vector, though overexpression of Rab26 does not alter the protein levels of Syt1, Syntaxin1, and SNAP25 (S4A Fig). However, expression of Rab26 had no effects on Syt1 binding to Syntaxin-1 (Fig 7C and 7D). Ca2+ stimulation promoted Syt1 binding to SNAP25, which was inhibited by Rab26 expression (Fig 7E and 7F). These results suggest that interaction of Rab26 with Syt1 inhibits Syt1 binding to SNAP25. Rab26 thus may restrict insulin secretion via sequestering Syt1 from interacting with SNAP-25. PPT PowerPoint slide

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TIFF original image Download: Fig 7. Rab26 influences the interaction between Syt1 and SNARE complex. (A) 293t cells were cotransfected myc-Vector and GFP-SNAP25 as control group, myc-Rab26 and GFP-SNAP25 as experimental group, incubated overnight with GST-Syt1 beads at 4°C through GST-pulldown experiment, washed by lysis buffer, and analyzed by western blotting with GFP antibody. (B) Quantitative analysis of the results of A from 3 independent experiments. (C) 293t cells were transfected myc-Vector and GFP-Syntaxin 1A as control group, myc-Rab26 and GFP-Syntaxin 1A as experimental group, incubated with GST-Syt1 beads, and analyzed by western blotting with GFP antibody. (D) Quantitative analysis of the results of C from 3 independent experiments. (A), (E) Cells were incubated for 5 min at 37°C in the presence of Ca2+ (10 μM CaCl2) or absence of Ca2+, indicating that Rab26 reduces the interaction between Syt1 and SNAP25. (F) Quantitative analysis of the results of E from 3 independent experiments. (n = 3; NS, not significant, ***P < 0.001, **P < 0.01, *P < 0.05, t tests). The numerical values that were used to generate graphs and histograms can be found in S1 Data. https://doi.org/10.1371/journal.pbio.3002142.g007 As Syt1 can bind to phospholipids, we investigated whether Rab26 interaction with Syt1 influences Syt1 binding to phospholipids. In vitro overlay assay revealed that Rab26 did not affect the interaction of Syt1 with PI(4,5)P 2 and PS (S4B Fig). Therefore, Rab26 inhibits insulin secretory granules exocytosis probably by interfering Syt1 interaction with SNARE complex and consequently inhibits insulin secretory granules fusion with the plasma membrane.

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[1] Url: https://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.3002142

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