## 3 Result
### Dynamic Glucose-Stimulated C-Peptide Secretion Profiles of Human Pls and HLSC-ILS
Similar to the previous protocol (SPGP), the kinetic curve of C-peptide secretion of PIs derived from healthy donors and HLSC-ILS (Figure 3B-left) were characterized by two main peaks of secretion during both the HG stimuli (HG\({}_{1}\) and HG\({}_{2}\)), and by a well-defined basal state during low-glucose concentrations (LG\({}_{1}\)-LG\({}_{2}\)-LG\({}_{3}\)). Nevertheless, the main difference between the two was in the amplitude of the responses (\(p\leq 0.05\)) with the healthy PIs being higher. Using this protocol, the dysregulated secretion profiles observed in PIs derived from obese donors did not substantially differ from those of healthy donors. Interestingly, the T2 DM profiles showed an overall lower amplitude of responses together with a late response to both HG\({}_{1}\) and LG\({}_{2}\) stimuli (Figure 3B-left). In particular, instead of responding to the actual stimuli, they showed a maximum (HG\(\rightarrow\)LG)\({}_{1}\) and minimum response during the transition states (LG\(\rightarrow\)HG)\({}_{2}\). The third protocol applied was the ARG, in which concentrations of glucose ranging from 3-30 mM were perfused and samples were collected every 10 min (Figure 2C). This protocol was performed to investigate glucose sensitivity, i.e., the concentration threshold which triggers the first C-peptide secretion response (defined as SI \(\geq\) 2). As shown in Figure 3C-left, this concentration ranged from 5-8 mM for human PIs independently of its clinical classification (healthy, obese, and T2 DM). Interestingly, the kinetic curve (but not the amplitude of response) in both obese and T2 DM groups were similar. They reached a peak of secretion between 12-15 mM followed by a sustained decrease until 30 mM of glucose (Figure 3C-left). Conversely, the healthy group showed a transient plateau of C-peptide secretion between 8-20 mM followed by a further increase between 23-28 mM of glucose (Figure 3C-left). In HLSC-ILS the glucose threshold for C-peptide secretion was 4 mM (earlier than in the other groups) and was followed by a reduction in secretion between 5-20 mM of glucose. A trend in restoration of C-peptide secretion was detected from 20-27 mM and a relative reduction at the end of perfusion (28-30 mM). Finally, the CHG protocol (Figure 2D) was designed to study the well-known biphasic pattern of insulin/C-peptide secretion in insulin-producing cells. It involves perfiusing 17 mM of glucose followed by sample collection every 5 min for 65 min. As expected, the secretion profile of healthy PIs was characterized by a rapid increase during the first 10 min of stimulation (first phase), followed by a decreased but sustained secretion of C-peptide (second phase) (Figure 3D-left). Interestingly, the obese group showed a significant (\(p\leq 0.05\)) delay in its first phase compared to the healthy group, while in the T2 DM group a virtual loss of the first phase was observed. Moreover, similar to our previous protocols, the amplitude of C-peptide secretion was higher in the obese group compared to T2 DM. On the other hand, HLSC-ILS displayed a robust first phase of secretion (SI-5) followed by an oscillatory second phase, characterized by peaks of secretion every 15 min (Figure 3D-left). To better compare the experimental groups (Table 1) and HLSC-ILS, the cumulative area under the curve (AUC) derived from each C-peptide secretion profile was analyzed (Figure 3A-D-right). By using this approach, we noticed that only in the SPGP protocol (Figure 3A-right) (high-glucose concentration: 28 mM), the AUC in the obese group was almost threefold higher compared to the other groups (Figure 3B-D-right). However, with this protocol, no significant differences were observed between the healthy and T2 DM groups. Conversely, in the DPG protocol (Figure 2B) the AUC of the C-peptide secretion profile in the healthy group was significantly higher than in the other conditions (obese, T2 DM, and HLSC-ILS). However, we did not find differences between obese and T2 DM on analyzing this protocol (Figure 3B-right).