## 4 Discussion
Butyrate is a physiological stimulus of GPCR, including GPR41 and GPR43. Using specific GPR41 and GPR43 inhibitors, we demonstrated that the increase of insulin expression may not be mediated through binding to one of these receptors. Since butyrate triggered beta cell development, it was surprising that GSIS was only modestly increased. Although we observed an upregulation of maturation markers (NKXe.1) and a downregulation of markers of immature beta cells (ALDOB), this may be explained by the fact that the beta cells are still not completely matured. Another explanation is that the binding of butyrate to GPR41 and GPR43 may influence GSIS differentially. GPR41 is coupled to Gi/o, which decreases intracellular cAMP levels by inhibition of adenylyl cyclase and thereby inhibiting PKA and EPAC mediated insulin secretion. GPR43 stimulation is linked with both Gi/o and Gq, decreasing cAMP levels and enhancing GSIS by increasing cytoplasmic calcium concentrations and activating PKA [20; 27; 28]. In primary isolated human islets, butyrate did not significantly stimulate GSIS. Insulin secretion of human islets were inhibited when treated with specific GPR43 agonists [29]. Therefore, it can be speculated that butyrate treatment increased beta cell maturation, but in parallel attenuated GSIS of NPICCs by binding to GPR43. Further experimental studies with specific blockers of the Gai and Gaq pathways are needed to determine how butyrate modulate regulation of insulin secretion in NPICCs. Another part of the physiological effects of butyrate is the modulation of chromatin acetylation and gene transcription by inhibition of class I and II histone deacetylases (HDAC), which regulate gene transcription by deacetylation of histones and transcription factors [30; 31]. In general, hyperacetylation is associated with increased gene expression while hypoacetylation favours gene silencing. Class I and class II HDAC are expressed in developing pancreas and in human and rat islets and are differentially regulated by metabolic and inflammatory mediators [32; 33]. It has been shown that acetylation of histone 4 is involved in glucose-dependent activation of the insulin gene promotor in the mouse beta cell line MIN6 and that PDX1 interacts with HDAC1 and HDAC2 to downregulated insulin expression at low glucose levels [34; 35]. In vivo studies reported on decreased beta cell apoptosis and increased beta cell proliferation through modification of histone acetylation in streptozotocin-diabetic mice after butyrate treatment [36]. We confirmed that butyrate inhibited histone deacetylase enzyme activity in NPICCs by using HDAC assays. Our study revealed that inhibition of class I HDAC, which includes HDAC1, HDAC2, HDAC3 and HDAC8, by specific small molecules such as mocetinostat strongly induced insulin expression but did not promote differentiation of alpha and delta cells in NPICCs and thereby mimicked the maturation effect induced by butyrate. Because class II HDACi MC1568 and TMP269 did not influence insulin expression our data suggest that class I HDACi specifically modify gene regions involved in porcine beta cell maturation. These findings are in contrast to previous studies in fetal rat pancreatic cells reporting on an upregulation of _NGN3_ gene expression and a strong increase of alpha cells and pancreatic polypeptide (PP) expressing cells by treatment with butyrate, TSA and MS275, respectively [32]. Class I specific HDACi, MS275 and valpro acid strongly decreased insulin and somatostatin expressing cells and treatment with the selective class II HDACi, MC1568, enhanced rat beta and delta cells [37]. In agreement with our results, exposure of fetal rat explants to TSA and butyrate strongly enhanced differentiation of insulin-expressing beta cells [32]. These data suggests that there may be major differences in the regulation of proendocrine transcription factors and insulin gene expression between rat and porcine pancreatic cells.