T2D-associated ARAP1 regulates GTPase activity, insulin processing and secretion in the pancreatic beta cell. J. R. Kulzer, R. C. McMullan, M. P. Fogarty, K. L. Mohlke Department of Genetics, UNC Chapel Hill, Chapel Hill, NC.
We previously proposed ARAP1 as a functional gene at a GWAS locus for type 2 diabetes (T2D) and fasting proinsulin, however, the biological mechanisms connecting ARAP1, proinsulin, and T2D remain unknown. We showed that the T2D-risk and proinsulin-decreasing rs11603334-C allele, located in an ARAP1 promoter, is associated with higher ARAP1 mRNA expression in human islets. ARAP1 protein contains both an ArfGAP and a RhoGAP domain, which catalyze inactivation of Arf and Rho family GTPases by facilitating GTP to GDP hydrolysis. These GTPases regulate Golgi transport, membrane trafficking, and actin cytoskeleton dynamics, processes involved in insulin granule packaging and trafficking. We hypothesized that ARAP1 may act through one or more Arf or Rho GTPases to exert a regulatory role on insulin processing and/or secretion. To identify which GTPases are regulated by ARAP1 in the beta cell, we measured GTPase activity following transient ARAP1 overexpression. Preliminary results show that exogenous overexpression of wild-type (wt) human ARAP1 in the mouse insulinoma cell line MIN6 led to a 58% decrease in Arf5-GTP levels and a 48% decrease in Arf1-GTP levels. Exogenous overexpression of an ARAP1 mutant with catalytically inactive ArfGAP and RhoGAP domains rescued levels of Arf1-GTP, but not Arf5-GTP. Using confocal fluorescence microscopy, we observed moderate colocalization of ARAP1 with Arf1 near the Golgi in MIN6 and in 832/13 rat insulinoma cells, and with Arf6 in dispersed human islet beta cells. To examine the effect of ARAP1 on proinsulin and insulin secretion, we exogenously overexpressed wt ARAP1 in MIN6 cells and measured supernatant insulin levels following KCl stimulation. We observed a 20% decrease in proinsulin secretion (P < .001), consistent in direction with GWAS findings that rs11603334-C is strongly associated with decreased plasma proinsulin levels. In the same samples, insulin secretion was found to be increased 70% (P < .03), suggesting a role for ARAP1 in proinsulin-to-insulin processing. ARAP1 ArfGAP/RhoGAP mutants rescued both proinsulin and insulin levels. Given the potential for species differences, we are extending these studies to human islets; we are also evaluating additional candidate GTPases. Our results suggest that upon acute stimulation, overexpressed ARAP1 may lead to decreased levels of secreted proinsulin through its regulation of Arf1 and/or Arf5 GTPase activity in pancreatic beta cells.
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