## 3 Results
### ZIP3 Transporters in the Plasma Membrane of SHED \(\beta\)-Cells Are Functional  
mRNA levels for Glucose transporter 2 (GLUT2) and Insulin precursor 1 (INS1) increased as SHED \(\beta\)-cells matured (Figure 3A). Supplementation with zinc significantly increased the expression of GLUT2 and INS1. Results of Western blot analysis indicated that ZIP3 transporters were present in the plasma membrane (Figure 3B). Using siRNA, we confirmed that ZIP3 transporters in the plasma membrane were functional. Results of ELISA assays for insulin levels and zinc contents indicated that siZIP3 partially reduced insulin secretion by SHED \(\beta\)-cells. Intracellular zinc concentrations showed a significant decrease in ZIP3 siRNA transfectants compared to SCR (scrambled) transfectants (Figure 3C). In the same culture, the amount of insulin secreted into the medium was also significantly reduced in the siZIP3 transfectants compared with the SCR transfectants (Figure 3D). These results suggest that insulin production by SHED \(\beta\)-cells may depend on the zinc concentration in the cytosol as well as the total amount of ZIP3 protein in the cells. Figure 3: Changes in mRNA levels of GLUT2 and INS1 as SHED cells mature into insulin-producing and -secreting SHED \(\beta\)-cells. Panel (**A**) shows mRNA expression of GLUT2 and INS1 at d10 in response to supplementation with 50 \(\upmu\)M zinc (n = 3, * \(p<0.01\)). (**B**) Western blot analysis of ZIP3 protein in membrane and cytosol of SHED \(\beta\)-cells. Arrowhead approximates the position of molecular weight (M.W.) of 34. Note the siRNA knocked down ZIP3 band expression on the right-most column for siZIP3 extracted from the membrane. MEK1/2 are ubiquitous kinase enzymes. Note that pan-Cadherin expresses in the membrane only. (**C**) Effects of ZIP3 knockdown on zinc levels compared with cells treated with scrambled (SCR) siRNA. (**D**) Effects of ZIP3 knockdown on insulin secretion compared with cells treated with scrambled (SCR) siRNA. * \(p<0.01\). Figure 2: Relative mRNA levels of GABAA receptors \(\upalpha\)1, \(\upalpha\)2, \(\upalpha\)3, and \(\upalpha\)6 (**A**) and ZIP3 transporters (**B**) relative to 18S ribosomal RNA in stem cells from human exfoliated deciduous teeth (SHED) cells as they mature into insulin-producing and -secreting SHED \(\beta\)-cells. * \(p<0.01\), ** \(p<0.0001\). Supplementation of Zinc during Differentiation Affects GABA-Induced Ca\({}^{2+}\) Responses in SHED \(\beta\)-Cells  
To investigate the effects of zinc on GABA-induced calcium signaling, we investigated the excitability of SHED \(\beta\)-cells using Ca\({}^{2+}\) imaging. We quantified the proportion of randomly selected SHED cells (n = 93) and SHED \(\beta\)-cells (n = 103) that responded to KCl and/or Ach. A change in the Fura signal (\(\Delta\)340/380) induced by the stimulus that exceeded the pre-stimulation level by \(>\)15% was counted as a positive response. We know that differentiation of \(\beta\)-cells from human embryonic stem cells is accompanied by a KCl-induced increase in intracellular free Ca\({}^{2+}\) concentration ([Ca\({}^{2+}\)]i), with enhanced expression of voltage-dependent Ca\({}^{2+}\) channels (VDCC) [24,25]. Therefore, differentiation of non-excitable SHED cells (Figure 4(Aa)) into SHED \(\beta\)-cells (Figure 4(Ab)) should be accompanied by functional expression of molecules responsible for Ca\({}^{2+}\) signaling. For ratiometric Ca\({}^{2+}\) imaging analysis on SHED and SHED \(\beta\)-cells using the Ca\({}^{2+}\)-sensitive fluorescent dye Fura-2 AM, we used KCl-induced increases in [Ca\({}^{2+}\)]i as a functional marker of excitable SHED \(\beta\)-cells. When undifferentiated SHED cells were exposed to Ach, approximately 25% of cells responded by increasing [Ca\({}^{2+}\)]i (Figure 4(Ac)). When the same SHED cells were exposed to KCl, only 2% of cells showed a noticeable increase in [Ca\({}^{2+}\)]i. In contrast, a majority of SHED \(\beta\)-cells (\(>\)60%) showed robust Ca\({}^{2+}\) responses to Ach (Figure 4(Ac), blue segments).