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Insulin plays a critical role in mammalian glucose homeostasis, and dysfunctional insulin secretion results in diabetes. Insulin maturation in pancreatic β cells can be generally divided into three stages: the first stage is the biosynthesis of proinsulin in the endoplasmic reticulum (ER), the second stage is the transport of proinsulin from the ER into the Golgi, and the third stage is the cleavage of proinsulin into insulin and C peptide in immature insulin granules (Dodson and Steiner, 1998). Previous studies reported that Rab family, which belongs to the small GTPase family, is involved in the regulation of insulin maturation. For example, Rab3, Rab11, Rab27, and Rab37 were associated with insulin-containing secretory granules and regulation of their exocytosis (Yi et al., 2002; Sugawara et al., 2009; Ljubicic et al., 2013; Cazares et al., 2014). A recent report showed that Rab2A can promote either insulin secretion or ER-associated degradation of proinsulin (Sugawara et al., 2014). By re-analyzing a dataset of microarray data derived from islets of type 2 diabetes patients (Dominguez et al., 2011), we found that rab1a mRNA expression was significantly decreased (P < 0.001, Fig. S1A). To confirm this observation, we compared the Rab1A protein levels in islets isolated from Wistar rats with those from diabetic Goto-Kakizaki (GK) rats that have spontaneous type 2 diabetes mellitus. Western blotting analysis showed that Rab1A expression was significantly reduced in GK islets (Fig. S1B). Taken together, Rab1A expression is down-regulated in diabetic islets, implying that Rab1A has an important function in pancreatic β-cells. We examined the sub-cellular localization of exogenous Rab1A in rat insulinoma INS-1E cells by using confocal microscopy. It has been reported that the Rab1A is mainly localized at the endoplasmic reticulum (ER)-Golgi membranes (Allan et al., 2000; Moyer et al., 2001). In agreement with previous studies, we found that Rab1A is mainly located in the cis-Golgi and endoplasmic reticulum (ER)-Golgi intermediate compartment (ER-GIC) in INS-1E cells (Fig. S2). To investigate the function of rab1a gene, we used the CRISPR/Cas9 approach to delete the rab1a gene in INS1E cells. Two cell clones (KO-2 and KO-3) were identified that contained additional T nucleotides in the rab1a gene, which led to a frameshift mutation (Fig. S3A). No detectable Rab1A was found in KO-2 or KO-3 cells by Western blotting (Fig. S3B) and RT-PCR (Fig. S3C). We examined glucose-stimulated insulin secretion (GSIS) in INS-1E cells with or without Rab1A function. The results showed that insulin secretion in KO-2 and KO-3 cells was significantly decreased compared with that in the control cells (Fig. 1A). Since the RT-PCR results showed no significant changes in insmRNA expression levels in KO-2 cells (Fig. 1B), we propose that Rab1A is not involved in regulating ins transcription. Western-blotting results showed that the proinsulin content of KO-2 cells was not significantly different from that of the control cells, but the matured insulin in KO-2 cells was largely decreased compared with that of the control cells (Fig. 1C). In addition, using rat insulin and proinsulin ELISA kits, we showed that rab1a knockout resulted in a significantly decreased insulin content (Fig. 1D, right panel), whereas no significant proinsulin content changes were detected (Fig. 1D, left panel). We next expressed wild type Rab1A and its mutants Q70L (a GTP-restricted mutant, activated form) and S25N (a GDP-restricted mutant, inactivated form) by lentivirus infection to see whether the activated form of Rab1A is essential to INS-1E cells’ function. We observed that overexpression of Rab1A WT or Rab1A Q70L resulted in no apparent increase of basal insulin secretion (BSIS), GSIS and insulin content. This lack of effect was not surprising, given that endogenous activated form of Rab1A may be functionally enough. But overexpression of Rab1A S25N significantly reduced BSIS, GSIS and insulin content (Fig. S4), suggesting that the activated form of Rab1A is essential to insulin secretion and insulin content. To further confirm that Rab1A regulates proinsulin to insulin conversion, we established KO + Rab1A cells by infecting KO-2 cells with a Rab1A-lentivirus (Fig. 1B and 1C). The exogenous expression of Rab1A in KO + Rab1A cells recovered the insulin content to levels that were similar to those observed in the control INS-1E cells (Fig. 1C and right

Rab1A mediates proinsulin to insulin conversion in β-cells by maintaining Golgi stability through interactions with golgin-84