Mouse iPSC Chemical Reprogramming Cocktails Kit plus br Mate

Materials and methods
Results
Discussion The present study confirms that DPP-4, a proteolytic enzyme that inactivates multiple substrates (including incretin hormones), is expressed in human pancreatic alpha Mouse iPSC Chemical Reprogramming Cocktails Kit plus (Omar et al., 2014, Bramswig et al., 2013). By performing electron microscopy immunogold analysis, we found that in ND islets around 90% of alpha cells are positive for DPP-4 (Fig. 2A). The intracellular localization of the enzyme has been shown previously by electron microscopy in pig alpha cells, mainly in association with the glucagon granules (Poulsen et al., 1993, Grondin et al., 1999). Importantly, the results of our present confocal and electron microscopy evaluations demonstrate that DPP-4 is also present in approximately 25% of insulin containing cells (Fig. 2A). Accordingly, we found that the enzyme is expressed in EndoC-βH1 cells, a human beta cell line shown to possess analogous physiological characteristics to primary human beta cells (Solimena et al., 2017, Gurgul-Convey et al., 2015, Andersson et al., 2015). In previous work, it has been observed that DPP-4 is also expressed in primary rodent beta cells and INS-1832/13 cells (Liu et al., 2014). Overall, these findings demonstrate that DPP-4 is expressed not only in human alpha, but also beta cells. Recent work performed with single islet cells has not reported the expression of DPP-4 in non-diabetic beta cells (Segerstolpe et al., 2016, Blodgett et al., 2015). Whether these discrepancies are due to beta cell heterogeneity, low grade DPP-4 expression in beta cells or differences in the techniques used (including the low coverage inherent to single cell RNAseq, that usually detects around 7–10.000 genes) remains to be elucidated. The pathophysiological role of DPP-4 in endocrine pancreatic islet cells is still uncertain, but several possibilities can be envisaged. Some substrates of DPP-4, such as peptide YY, are expressed in the embryonic pancreas and may play a role in endocrine cell differentiation (Jackerott and Larsson, 1997, Lukinius et al., 1992). In addition, DPP-4 mediates cellular adhesion by its interaction with fibronectin, which is also involved in organ development and organization (Shimomura et al., 2006). Furthermore, since islet cells (in particular alpha cells) are known to produce GLP-1 under certain circumstances (Marchetti et al., 2012, Ellingsgaard et al., 2011), DPP-4 might contribute to modulate intra-islet GLP-1 concentrations. Interestingly, we observed that T2D islet cells have lower DPP-4 expression, as compared to ND cells (Fig. 1, Fig. 2). Previously, a reduced activity of this peptidase was observed in human T2D islet lysates (Omar et al., 2014). As mentioned above, GLP-1 is also produced by islet cells (alpha cells in particular) (Marchetti et al., 2012, Ellingsgaard et al., 2011). Given the beneficial effect of GLP-1 on beta cells (Campbell and Drucker, 2013, Seghieri et al., 2013, Vilsbøll et al., 2003, Nauck and Meier, 2016, Ahrén et al., 2002, Chia and Egan, 2008, Muscelli et al., 2012, Dalle et al., 2013, Puddu et al., 2013), one might speculate that reduced expression and activity of DPP-4 represents an attempt to protect beta cells under stressful conditions by increasing local active GLP-1 concentrations. Accordingly, the use of DPP-4 inhibitors has been shown to exert direct beneficial effects on beta cell function and survival. Previous works demonstrated that linagliptin protected isolated ND human islets from the damage induced by elevated glucose, palmitate, cytokines or H2O2 (Shah et al., 2013). In the present study, we confirm that a different DPP-4 inhibitor, MK-0626, directly prevented beta cell death and dysfunction caused by cytokines in both primary human islets and the human cell line EndoC-βH1 (Fig. 3 and Supplemental Fig. 2). We tested cytokines since islet inflammation can play a role in both type 1 and type 2 diabetes development and progression (Eizirik et al., 2012b, Marchetti, 2016). Of more importance, our data showed that DPP-4 inhibition directly improves function, survival and ultrastructure of human diabetic beta cells (Fig. 4). It has been previously shown that cytokine exposure does not apparently affect the expression of DPP-4 in isolated human islets (Eizirik et al., 2012a). Since DPP-4 activity may change independent from its expression (Omar et al., 2014), the beneficial effects of DPP-4 inhibition may not be necessarily accompanied by changes in the expression of the enzyme.