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  • A localization of GK to


    A localization of GK to both a cytoplasmic and perinuclear area of normal rat pancreatic beta-cells was reported in 1996 (Noma et al., 1996), which changed on acute hyperglycemia to a more diffuse staining throughout the cytoplasm. The localization of GK within the nucleus was first reported by Murata et al. (Murata et al., 1997), in the mouse-derived insulinoma cell line MIN6. They detected GK immunoreactivity both in the nucleus and in the cytoplasm, with the strongest signal in the nucleus. The mechanism governing the nuclear import of GK, however, has never been reported. Posttranslational modification of proteins by members of the small ubiquitin-like modifier (SUMO) family modulates and regulates diverse cellular processes. Among the most frequent and thoroughly studied effects are protein stability, nucleocytoplasmic trafficking, and transcriptional regulation. We recently reported that GK is modified by SUMOylation in vitro, in both rat insulinoma SU 5402 australia (INS-1) and MIN6 cells (Aukrust et al., 2013). Compared to the liver isoform of human GK (hGK) the pancreatic isoform of the human enzyme was the preferred substrate for SUMOylation. Moreover, this posttranslational modification increased the cellular stability and activity of pancreatic hGK, and the E3 SUMO ligase and nuclear pore protein RanBP2 increased the level of GK SUMOylation in vitro. Here, we continue our studies on the nuclear localization of GK in the MIN6 cell line as well as in human and mouse isolated islets, and in beta-cells of human and mouse pancreatic sections. Since SUMOylation is known to regulate/modulate nucleocytoplasmic trafficking (Lin et al., 2003, Sehat et al., 2010, Sobko et al., 2002, Wood et al., 2003), and given our recent findings that the function of pancreatic GK is regulated by SUMOylation, we hypothesized that SUMOylation could affect the nuclear localization of this enzyme.
    Material and methods
    Discussion GK plays a key role in stimulus-secretion coupling in pancreatic beta–cells, and the subcellular compartmentation of the enzyme has been a target for many investigations (Lenzen, 2014). In several studies, immunostaining of pancreatic GK has shown a marked subcellular heterogeneity, with an apparent uniform distribution in the cytoplasm (Jetton and Magnuson, 1992, Jorns et al., 1999) and an association with various subcellular structures including insulin granules (Toyoda et al., 1999), actin filaments (Murata et al., 1997), and mitochondria (Danial et al., 2003, Liu et al., 2009). In contrast to hepatocytes (Agius et al., 1996, de la Iglesia et al., 1999), a nuclear localization of GK in beta–cells has been more controversial. The first report on a near-nuclear association of GK was based on immunostaining of the rat pancreas (Noma et al., 1996). Later, Murata et al. followed up by demonstrating GK localization within beta–cell nuclei of MIN6 cells (Murata et al., 1997). More recently, a study in MIN6 cells demonstrated an increased ratio of several transcripts in the nucleus compared to the cytoplasm, of which GK was one of the predominant ones (Bahar Halpern et al., 2015). By using different cellular techniques, and a variety of GK-targeted antibodies, we here confirm the nuclear localization of GK in MIN6 cells. The level of nuclear compared to cytosolic overexpressed GK in MIN6 cells was substantially lower than the ratios reported for GK in hepatocytes at high glucose concentration (Mukhtar et al., 1999). Moreover, we demonstrate, for the first time, that GK is present in nuclear fractions from both human and mouse islets. In general, the ratio of nuclear versus cytosolic GK was higher than in MIN6 cells, however, still lower than what reported in hepatocytes. Furthermore, we also detect GK in the nucleus of human beta-cells by immunostaining of pancreatic sections. In the human beta–cells, GK localized in part with DAPI (nucleus) and in part with insulin (cytoplasm). Co-localization of GK with insulin granules has previously been reported in rat beta–cells (Toyoda et al., 1999). In mouse endocrine pancreas, however, the GK staining was more restricted to the nucleus.