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. 2022 Mar 1;11(3):388.
doi: 10.3390/biology11030388.

EXOC6 (Exocyst Complex Component 6) Is Associated with the Risk of Type 2 Diabetes and Pancreatic β-Cell Dysfunction

Nabil Sulaiman  1 , Mahmood Yaseen Hachim  2 , Anila Khalique  3 , Abdul Khader Mohammed  3 , Saba Al Heialy  2 , Jalal Taneera  3   4
Affiliations

EXOC6 (Exocyst Complex Component 6) Is Associated with the Risk of Type 2 Diabetes and Pancreatic β-Cell Dysfunction

Nabil Sulaiman et al. Biology (Basel). .

Abstract

EXOC6 and EXOC6B (EXOC6/6B) components of the exocyst complex are involved in the secretory granule docking. Recently, EXOC6/6B were anticipated as a molecular link between dysfunctional pancreatic islets and ciliated lung epithelium, making diabetic patients more prone to severe SARS-CoV-2 complications. However, the exact role of EXOC6/6B in pancreatic β-cell function and risk of T2D is not fully understood. Herein, microarray and RNA-sequencing (RNA-seq) expression data demonstrated the expression of EXOC6/6B in human pancreatic islets. Expression of EXOC6/6B was not affected by diabetes status. Exploration of the using the translational human pancreatic islet genotype tissue-expression resource portal (TIGER) revealed three genetic variants (rs947591, rs2488071 and rs2488073) in the EXOC6 gene that were associated (p < 2.5 × 10−20) with the risk of T2D. Exoc6/6b silencing in rat pancreatic β-cells (INS1-832/13) impaired insulin secretion, insulin content, exocytosis machinery and glucose uptake without cytotoxic effect. A significant decrease in the expression Ins1, Ins1, Pdx1, Glut2 and Vamp2 was observed in Exoc6/6b-silenced cells at the mRNA and protein levels. However, NeuroD1, Gck and InsR were not influenced compared to the negative control. In conclusion, our data propose that EXOC6/6B are crucial regulators for insulin secretion and exocytosis machinery in β-cells. This study identified several genetic variants in EXOC6 associated with the risk of T2D. Therefore, EXOC6/6B could provide a new potential target for therapy development or early biomarkers for T2D.

Keywords: EXCO6; INS-1 cells; RNA sequencing; insulin secretion; siRNA silencing; type 2 diabetes.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Expression of EXOC6/6b genes in human pancreatic islets. (A) Microarray mean expression of KCNJ11, GLUT1, EXOC6 and EXOC6B in nondiabetic human islets (n = 45). (B) RNA sequencing expression of KCNJ11, GLUT1, EXOC6 and EXOC6B in nondiabetic human islets (n = 50). (C) Differential expression analysis (RNA-seq) of EXOC6 and EXOC6B in human islets obtained from diabetic/hyperglycemic donors (n = 27) compared to nondiabetic/normoglycemic donors (n = 50). ***; p < 0.001. n.s.; not significant. Bars represent mean  ±  SD.
Figure 2
Figure 2
Silencing of Exoc6/6b in INS-1 cells. (A) Expression analysis of Exoc6 and Exoc6b as determined by qPCR expression 48 h post-transfection. (B) Percentage of cell viability evaluated by MTT assay in Exoc6/6b-silenced cells and control cells. (C) Apoptosis level in Exoc6/6b-silenced INS-1 cells compared to negative control cells determined by flow cytometry analysis. (D–E) qPCR expression analysis of exocyst subunits in Exoc6 (D) or Exoc6b-silenced cells (E). ***; p < 0.001. n.s.; not significant. Bars represent mean ± SD. Data were acquired from 3 different experiments.
Figure 3
Figure 3
Impact of Exoc6/6b silencing on insulin release in INS-1 cells. (A,B) Measurement of insulin secretion (normalized to cellular protein content) stimulated with 2.8 mM glucose, 16.7 mM glucose, or 35 mM KCl + 2.8 mM glucose in Exoc6 (A) or Exoc6b (B) silenced cells compared to control cells. (C) Insulin content measurements normalized to protein content in Exoc6/6b-silenced cells compared to control cells. (D) assessment of ROS levels by fluorescence intensity in Exoc6/6b-silenced INS-1 cells compared to control cells. (E) Determination of glucose uptake in Exoc6/6b-silenced cells compared to control cells. *; p < 0.05. **; p < 0.01. ***; p < 0.001. ns.; not significant. Bars represent mean ± SD. Data are acquired from 3 different experiments.
Figure 4
Figure 4
Impact of Exoc6/6b silencing on the expression of β-cell function genes. Total mRNA/protein was extracted from Exoc6/6b-silenced cells or siRNA negative control after 48 h of transfection and subjected for qPCR or western blot analysis. qPCR expression analysis of Ins1, Ins2, Pdx1, NeuroD1, Gck, Glut2, InsR and Vamp2 in Exoc6-silenced cells (A) or Exoc6b-silenced cells (B) compared to control cells. Western blot analysis of Pro/insulin, PDX1, NEUROD1, PDX1, GLUT2, GCK, INSβ and VAMP2 relative to the endogenous control protein β-actin in Exoc6-silenced cells (C) or Exoc6b-silenced cells (D). Data were obtained from three independent experiments (Supplementary Figures S1–S3). *; p < 0.05. **; p < 0.01. n.s.; not significant. Bars represent mean  ±  SD.
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