Slets were pre-cultured for 48h and then exposed to 2.8 mM glucose for 1 h (basal), to 2.8 mM glucose including the adipocytokines for 1 h (basal+adipokine) and another subsequent hour to 16.7 mM glucose including the adipocytokines (stim+adipokine). The stimulatory index was calculated (F). (G, H) Islets were pre-cultured for 48 h and then exposed to 2.8 mM glucose for 1 h (basal), to 16.7 mM glucose for 1 h (stimulated) and another subsequent hour to 16.7 mM glucose including the adipocytokines (stim+adipokine). The stimulatory index was calculated (H). (I) Stimulatory index from human islets exposed to 2.8 mM glucose (basal) and subsequently to 1 h exposure to IBMX (100 mM)/Forskolin (10 mM) with or without Nampt or NMN was calculated. Results are means 6SEM from triplicates from three independent experiments from three donors. *p,0.05 to the respective untreated control, +p,0.05 to 2.8 mM basal glucose. doi:10.1371/journal.pone.0054106.gNampt and NMN Potentiate Glucose Stimulated Insulin Secretion in Human IsletsSince Nampt and NMN failed to protect human islets from apoptosis induced by diabetogenic conditions, we tested whether it may influence insulin secretion under basal conditions in culture. Human islets were chronically exposed to Nampt or NMN at 5.5 mM glucose for 72 h and GSIS was analysed thereafter. Nampt and its enzymatic product NMN did not influence beta-cell insulin secretion upon chronic exposure (Fig. 3A,B). Next, we 298690-60-5 investigated whether Nampt and NMN have an effect on longterm glucolipotoxicity and cytokine toxicity, induced by 72 h exposure of human islets to the mixture of 22.2 mM glucose and 0.5 mM palmitate or by the mixture of the cytokines IL-1b and IFN-c. Glucose stimulated insulin secretion was determined at the end of the 72 h culture. Glucose/palmitate as well as the cytokine mixture severely reduced the stimulatory index (Fig. 3C,D; 3.8and 1.8-fold respectively, p,0.05). Neither Nampt nor NMN changed GSIS in any of the conditions (Fig. 3C,D). This is in line with the above KDM5A-IN-1 chemical information described lack of influence of Nampt and NMN on beta-cell survival (Fig. 2). To determine the acute effect of Nampt and NMN on insulin secretion, we cultured the islets in the presence of the adipocytokine at low and high glucose concentrations for 1 h, respectively. At low glucose, Nampt and NMN elicited no significant effect on insulin secretion when compared to low glucose alone (Fig. 3E, basal +adipokine vs. basal). At high glucose conditions the GSIS was improved by Nampt and NMN. While glucose alone induced a 2.4-fold induction of insulin secretion, this induction was 2.0- and 1.8-fold induced by NMN and Nampt, respectively (p,0.05, Fig. 3E,F, stim+adipokine vs. basal), whencompared to 16.7 mM glucose alone. To exclude exhaustive effects on beta-cell insulin secretion, which could have occurred after stimulation with high glucose concentrations, we repeated the experiment by testing adipocytokine effects only at 16402044 high glucose conditions. Again, human islets were pre-cultured for 2 days at 5.5 mM glucose, basal glucose of 2.8 mM (Fig. 3G, basal) was added for 1 h followed by 1 h exposure to high glucose (16.7 mM) (Fig. 3G, stimulated) and then to high glucose in the presence of Nampt or NMN (Fig. 3G, stim+adipokine). All islets showed similar GSIS before the addition of the adipocytokine (Fig. 3G, stimulated). In contrast, islets which were 26001275 stimulated a 2nd subsequent hour with 16.7 mM glucose alone showed a decrease in GSIS (Fig.Slets were pre-cultured for 48h and then exposed to 2.8 mM glucose for 1 h (basal), to 2.8 mM glucose including the adipocytokines for 1 h (basal+adipokine) and another subsequent hour to 16.7 mM glucose including the adipocytokines (stim+adipokine). The stimulatory index was calculated (F). (G, H) Islets were pre-cultured for 48 h and then exposed to 2.8 mM glucose for 1 h (basal), to 16.7 mM glucose for 1 h (stimulated) and another subsequent hour to 16.7 mM glucose including the adipocytokines (stim+adipokine). The stimulatory index was calculated (H). (I) Stimulatory index from human islets exposed to 2.8 mM glucose (basal) and subsequently to 1 h exposure to IBMX (100 mM)/Forskolin (10 mM) with or without Nampt or NMN was calculated. Results are means 6SEM from triplicates from three independent experiments from three donors. *p,0.05 to the respective untreated control, +p,0.05 to 2.8 mM basal glucose. doi:10.1371/journal.pone.0054106.gNampt and NMN Potentiate Glucose Stimulated Insulin Secretion in Human IsletsSince Nampt and NMN failed to protect human islets from apoptosis induced by diabetogenic conditions, we tested whether it may influence insulin secretion under basal conditions in culture. Human islets were chronically exposed to Nampt or NMN at 5.5 mM glucose for 72 h and GSIS was analysed thereafter. Nampt and its enzymatic product NMN did not influence beta-cell insulin secretion upon chronic exposure (Fig. 3A,B). Next, we investigated whether Nampt and NMN have an effect on longterm glucolipotoxicity and cytokine toxicity, induced by 72 h exposure of human islets to the mixture of 22.2 mM glucose and 0.5 mM palmitate or by the mixture of the cytokines IL-1b and IFN-c. Glucose stimulated insulin secretion was determined at the end of the 72 h culture. Glucose/palmitate as well as the cytokine mixture severely reduced the stimulatory index (Fig. 3C,D; 3.8and 1.8-fold respectively, p,0.05). Neither Nampt nor NMN changed GSIS in any of the conditions (Fig. 3C,D). This is in line with the above described lack of influence of Nampt and NMN on beta-cell survival (Fig. 2). To determine the acute effect of Nampt and NMN on insulin secretion, we cultured the islets in the presence of the adipocytokine at low and high glucose concentrations for 1 h, respectively. At low glucose, Nampt and NMN elicited no significant effect on insulin secretion when compared to low glucose alone (Fig. 3E, basal +adipokine vs. basal). At high glucose conditions the GSIS was improved by Nampt and NMN. While glucose alone induced a 2.4-fold induction of insulin secretion, this induction was 2.0- and 1.8-fold induced by NMN and Nampt, respectively (p,0.05, Fig. 3E,F, stim+adipokine vs. basal), whencompared to 16.7 mM glucose alone. To exclude exhaustive effects on beta-cell insulin secretion, which could have occurred after stimulation with high glucose concentrations, we repeated the experiment by testing adipocytokine effects only at 16402044 high glucose conditions. Again, human islets were pre-cultured for 2 days at 5.5 mM glucose, basal glucose of 2.8 mM (Fig. 3G, basal) was added for 1 h followed by 1 h exposure to high glucose (16.7 mM) (Fig. 3G, stimulated) and then to high glucose in the presence of Nampt or NMN (Fig. 3G, stim+adipokine). All islets showed similar GSIS before the addition of the adipocytokine (Fig. 3G, stimulated). In contrast, islets which were 26001275 stimulated a 2nd subsequent hour with 16.7 mM glucose alone showed a decrease in GSIS (Fig.
