AGI-1067, a novel antioxidant and anti-inflammatory agent, enhances insulin release and protects mouse islets

Abstract

The antioxidant and anti-inflammatory compound AGI-1067 (succinobucol) has potential as an oral anti-diabetic agent. AGI-1067 reduces H(b)A1c, improves fasting plasma glucose, and reduces new-onset diabetes. We investigated AGI-1067 for possible effects on mouse pancreatic islets in vitro. Pretreatment with 10 microM AGI-1067 increased glucose-stimulated insulin secretion (11 mM) without affecting secretion in basal (3 mM) glucose. AGI-1067 enhanced the intracellular calcium response to glucose stimulation in 7 mM and 11 mM glucose, but had no effect in 28 mM or basal glucose. AGI-1067-pretreated islets also showed enhanced calcium responses to methyl pyruvate and alpha-ketoisocaproate at low doses, but not high doses. The AGI-1067-mediated effects on glucose-stimulated calcium were maintained during continuous diazoxide exposure, suggesting effects on the K(ATP)-channel-independent pathway. AGI-1067 also reduced cytokine-induced islet cell death and expression of iNOS, a key component in cytokine signaling. This is the first report of direct stimulatory and protective effects of a first-in-class potential anti-diabetic agent on pancreatic islets.

Figure 1

No acute effects of AGI-1067 on [Ca²⁺]_i in islets. (A) [Ca²⁺]_i was recorded for 3 min in 3 mM glucose, and then islets were treated with either 10 μM AGI-1067 (upper panel) or 100 μM tolbutamide (lower panel) for 5 min. [Ca²⁺]_i did not significantly change during AGI-1067 treatment (p=0.20, n=23), whereas a very large response to tolbutamide was observed (p<0.001, n=16). (B) Ca²⁺]_i was recorded for 10 min in 11 mM glucose (only 5 min are displayed), and then islets were treated with either 10 μM AGI-1067 (upper panel) or 100 μM tolbutamide (lower panel). AGI-1067 treatment did not appreciably change oscillatory patterns (n=11), whereas tolbutamide raised [Ca²⁺]_i and eliminated oscillations (n=22).

Figure 2

CTR shows no spectral overlap with fura-2 AM and does not interfere with islet physiological function. (A) Brightfield, (B) Image of CTR vs. unlabeled islets with 535 nm excitation (Ex) and 630 nm emission (Em). (C-D) Unlabeled (C, n=7) and CTR-labeled islets (D, n=6) showed similar [Ca²⁺]_i responses to glucose stimulation (3 to 28 mM). (E) No differences in [Ca²⁺]_i were observed during the glucose stimulation tests involving >100 islets in total. (F) CTR-labeling does not interfere with glucose-stimulated insulin secretion (n=6 sets of 50 islets per treatment group).

Figure 3

AGI-1067 augments GSCa and GSIS. (A) Representative examples of control (solid, n=12 islets) and AGI-1067-treated islet (dashed, n=10 islets). (B) Comparisons of mean [Ca²⁺]_i response for control or AGI-pretreated islets during phase 0 (ER dip), phase 1, and phase 2 of glucose stimulation. A total of 6 trials were preformed using n=37 control and 27 AGI-pretreated islets. (C) Insulin secretion from islets pretreated with 0, 5, or 10 μM AGI-1067. AGI-1067 significantly increased insulin secretion in 11 mM glucose, but not basal (3 mM) glucose (n=9 replicates using islets from 12 mice, *p<0.05, ***p<0.001).

Figure 4

Effects of AGI-1067 on glucose sensitivity. Islets were pretreated with 10 μM AGI-1067 or vehicle (control), and GSCa was recorded using stimulation of 7 mM glucose (A), 11 mM glucose (B), or 28 mM glucose (C). AGI-1067-pretreated islets showed a reduced phase 0 dip and an enhanced 1st phase response compared to the control group of islets in 7 mM glucose; a reduced ER dip and an elevated 2nd phase response compared to the control group in 11 mM glucose; no significant differences in 28 mM glucose. Traces in each panel consist of mean values of n=7 islets or more for each treatment. At least two trials were performed for each concentration of glucose with total islets numbers listed in Table 1.

Figure 5

Effects of AGI-1067 on islet stimulation by alternative fuels. (A-B) Islets were pretreated with 10 μM AGI or vehicle (control) and then recorded for [Ca²⁺]_i changes in response to stimulation by alpha-ketoisocaproate (KIC) or methyl pyruvate. (A-B) AGI-pretreated islets (dashed) showed a greater response to 2 mM KIC than untreated control (solid) islets (A), but no differences were observed with 10 mM KIC (B). (C-D) AGI-pretreated islets (dashed) also showed a greater response to 4 mM methyl pyruvate than untreated control (solid) islets (C), but no differences were observed with 20 mM methyl pyruvate (D). Data represent 1 of 3 trials that were conducted for each fuel. Mean values among combined trials and total islets numbers are shown in Table 1.

Figure 6

Effects of AGI-1067 on KCl-stimulated [Ca²⁺]_i response. (A) Islets pretreated with 10 μM AGI-1067 showed a reduced response to KCl stimulation compared to the control islets. (B) Mean [Ca²⁺]_i values before, during, and after KCl treatment. *p<0.05. A total of 6 trials were preformed using n=29 control and n=35 AGI-pretreated islets.

Figure 7

AGI-1067 pretreatment enhances GSCa in the presence of diazoxide. (A) Examples of GSCa for control (solid, n=10) and AGI-pretreated (dashed, n=11) islets in the presence of 250 uM diazoxide (a KATP channel opener) throughout the recording. Due to the presence of diazoxide the normal calcium response to glucose is disrupted. Traces are representative of data from 1 of 3 trials. (B) Mean values for basal [Ca²⁺]_i in 3 mM glucose + diazoxide, ER dip (phase 0), and [Ca²⁺]_i response to stimulation. Control (white, n=33) and AGI-1067-pretreated (black, n=35) islets differed markedly in their responses. Data are combined values from 3 separate trials. **p<0.01, ***p<0.001,

Figure 8

AGI-1067 protects against cytokine-induced cell death. (A-C) Images of islets (left panel) and their respective cell death rates indicated by PI fluorescence (right panel). Control islets (A) showed very low PI fluorescence compared to cytokine-treated islets (B); PI fluorescence was reduced but not completely eliminated among islets pretreated with 10 μM AGI-1067 and then exposed to cytokines (C). (D) Mean PI values in arbitrary units (a.u.) for each condition taken from 3 separate trials (n=33-40 islets per condition). *p < 0.01.

Figure 9

AGI-1067 effects on islet gene expression. (A) Insulin secretion from islets in 11 mM glucose following overnight treatment with pro-inflammatory cytokines and/or 10 μM AGI-1067. (B) Gene expression differences between control and AGI-1067-treated islets. (C) Gene expression for islets treated with cytokines alone and islets treated with AGI-1067 prior to overnight cytokine treatment. Fold differences are compared to expression levels in control islets as shown in (B). 50 islets were used for each replicate of each condition (n=6 replicates, *p<0.05).