## 3 Results
### Butyrate Enhances Protein Expression of Beta Cell Maturation Markers  
To further explore the effects of butyrate, NPICCs were analyzed by FACS before butyrate treatment and after exposure to 1000 uM butyrate for six days. As illustrated in Figure 2A-C, butyrate treatment significantly increased the number (43.8 \(\pm\) 4.7% vs. 33.1 \(\pm\) 3.0%) and the median fluorescence intensity of insulin positive cells compared to cells cultured in B-IC medium (_p_ < 0.01). Incubation with butyrate also resulted in a higher proportion of mature beta cells, defined by insulin+ and Nkx6.1+ co-staining (33.4 \(\pm\) 8.3% vs. 21.0 \(\pm\) 8.0%). This effect was not mediated by increased cell proliferation, since analysis by immunohistochemistry did not detect differences in Edu-positive cells in the presence or absence of butyrate (Supplemental Figure S2). Interestingly, the percentage of glucagon positive cells slightly decreased in butyrate treated cells (12.8 \(\pm\) 5.0% vs. 18.6 \(\pm\) 7.0%), whereas the number of somatostatin positive cells (10.1 \(\pm\) 1.0% vs. 10.0 \(\pm\) 2.7%) and Pdx1 positive cells (39.3 \(\pm\) 6.1% vs. 43.2 \(\pm\) 9.1%) was not altered.  
Although there was a significant upregulation of insulin gene and protein expression, insulin content (51.7 vs. 29.6 uU/ng DNA) and GSIS was only moderately increased from 1.7 to 2.1 (Figure 2F) suggesting that NPICCs still have not acquired a physiological glucose response.  
Figure 1: Exposure to butyrate significantly promotes beta cell maturation in a- and time-dependent manner. Relative gene expression of insulin (_INS_) (**A**,**D**), glucagon (_GCG_) (**B**,**E**), pancreatic and duodenal homeobox 1 (_PDX1_) (**C**,**F**), NK6 homeobox 1 (_NKX6.1_) (**G**), neurogeni 3 (_NGN3_) (**H**), and aldolase fructose-bisphosphate B (_ALDOB_) (**I**) assessed by RT-qPCR in NPICCs. (**A–C**) NPICCs were treated with 100–2000 \(\upmu\)M butyrate for six days, (**D–F**) 1000 \(\upmu\)M butyrate for one to eight days and (**G–I**) 1000 \(\upmu\)M butyrate for six days. Cells incubated without butyrate were used as controls. Data are presented as mean \(\pm\) SD of three to six independent experiments. * \(p<0.05\), ** \(p<0.01\), *** \(p<0.001\) and *** \(p<0.0001\) vs. control groups.  
Figure 2: Butyrate increased the number of insulin positive cells and insulin content. **(A)** FACS analysis of NPICCs before and after treatment with butyrate (1000 \(\upmu\)M) for six days. NPICCs were dispersed into single cells and analyzed by intracytoplasmic staining for insulin (AF647), glucagon (PE), Pdx1 (AF488) and Nkx6.1 (AF647). **(B,C)** Butyrate increased the number of insulin positive cells and the median fluorescence intensity (MFI) of insulin stained cells. **(D,E)** Immunofluorescence staining for insulin (red) and glucagon (green) confirmed increase of insulin positive NPICCs in the butyrate treated group (1000 \(\upmu\)M for six days). scale bars = 100 \(\upmu\)m. **(F)** GSIS and Insulin content were moderately increased in NPICCs treated with butyrate. Data are presented as mean \(\pm\) SD **(B,E,F)** or median \(\pm\) SD **(C)** of three to six independent experiments. **\({}^{**}p<0.01\) vs. control group.