Abstract
ABSTRACTWe previously reported that phospholipase C-related catalytically inactive protein (PRIP)-knockout mice exhibited hyperinsulinemia. Here, we investigated the role of PRIP in insulin granule exocytosis using Prip-knockdown mouse insulinoma (MIN6) cells. Insulin release from Prip-knockdown MIN6 cells was higher than that from control cells, and Prip knockdown facilitated movement of GFP-phogrin-labeled insulin secretory vesicles. Double-immunofluorescent staining and density step-gradient analyses showed that the KIF5B motor protein co-localized with insulin vesicles in Prip-knockdown MIN6 cells. Knockdown of GABAA-receptor-associated protein (GABARAP), a microtubule-associated PRIP-binding partner, by Gabarap silencing in MIN6 cells reduced the co-localization of insulin vesicles with KIF5B and the movement of vesicles, resulting in decreased insulin secretion. However, the co-localization of KIF5B with microtubules was not altered in Prip- and Gabarap-knockdown cells. The presence of unbound GABARAP, freed either by an interference peptide or by Prip silencing, in MIN6 cells enhanced the co-localization of insulin vesicles with microtubules and promoted vesicle mobility. Taken together, these data demonstrate that PRIP and GABARAP function in a complex to regulate KIF5B-mediated insulin secretion, providing new insights into insulin exocytic mechanisms.
Highlights
Insulin secretion from pancreatic b-cells is a highly dynamic process regulated by multiple stimuli, including nutrients, hormones, and neuronal inputs
phospholipase C-related catalytically inactive protein (PRIP) regulates neuronal surface expression of c subunit-containing GABAA receptors by controlling GABAA-receptor-associated protein (GABARAP) function (Mizokami et al, 2007). These findings suggest that PRIP and/or GABARAP may participate in the transport of insulin vesicles
We demonstrated that PRIP is a novel modulator of vesicle–kinesin complex formation
Summary
Insulin secretion from pancreatic b-cells is a highly dynamic process regulated by multiple stimuli, including nutrients, hormones, and neuronal inputs. Stimulation by glucose induces a biphasic pattern of insulin release. First-phase insulin release occurs within a few minutes after exposure to elevated glucose, and it is followed by a more sustained second phase (Rorsman et al, 2000). The first phase, during which insulin granules that. Second phase release correlates with mobilization of insulin-containing granules from the releasable pool into the cell periphery, which is mediated by microtubules. Insulin-containing cargo transport is regulated by the motor protein kinesin-1/KIF5 (Balczon et al, 1992; Cui et al, 2011; Meng et al, 1997; Varadi et al, 2002; Varadi et al, 2003)
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