Imeglimin amplifies glucose-stimulated insulin release from diabetic islets via a distinct mechanism of action

Abstract

Pancreatic islet β-cell dysfunction is characterized by defective glucose-stimulated insulin secretion (GSIS) and is a predominant component of the pathophysiology of diabetes. Imeglimin, a novel first-in-class small molecule tetrahydrotriazine drug candidate, improves glycemia and GSIS in preclinical models and clinical trials in patients with Type 2 diabetes; however, the mechanism by which it restores β-cell function is unknown. Here, we show that imeglimin acutely and directly amplifies GSIS in islets isolated from rodents with Type 2 diabetes via a mode of action that is distinct from other known therapeutic approaches. The underlying mechanism involves increases in the cellular nicotinamide adenine dinucleotide (NAD+) pool-potentially via the salvage pathway and induction of nicotinamide phosphoribosyltransferase (NAMPT) along with augmentation of glucose-induced ATP levels. Further, additional results suggest that NAD+ conversion to a second messenger, cyclic ADP ribose (cADPR), via ADP ribosyl cyclase/cADPR hydrolase (CD38) is required for imeglimin's effects in islets, thus representing a potential link between increased NAD+ and enhanced glucose-induced Ca2+ mobilization which-in turn-is known to drive insulin granule exocytosis. Collectively, these findings implicate a novel mode of action for imeglimin that explains its ability to effectively restore-β-cell function and provides for a new approach to treat patients suffering from Type 2 diabetes.

Fig 1. Imeglimin amplifies insulin secretion in islets from N0STZ rats.

Wistar Rat Islets (A) vs. N0STZ Rat Islets (B). Islets from N0STZ or healthy Wistar rats were incubated in the presence of 2.8 mM or 16.7 mM glucose. Insulin levels were measured in supernatants after 30 min of incubation. **p<0.01, ***p<0.001 vs. respective low glucose values; mean ± SEM; n = 6 wells with 6 islets per well. Effect of imeglimin and GLP1 on Insulin Secretion from N0STZ Rat Islets (C). Islets from N0STZ rats were incubated in the presence of 2.8 mM or 16.7 mM glucose with or without the tested concentrations of imeglimin or GLP1 10⁻⁷ M. Insulin levels were measured in supernatants after 30 min of incubation. The effect of imeglimin at 100 μM was significant, *p<0.05, vs. high glucose alone; mean ± SEM; n = 9–10 wells with 6 islets per well (note that when using an unpaired Student t-test, GLP1 also achieved statistical significance, p = 0.0054).

Fig 2. Imeglimin amplifies insulin secretion in islets from GK rats.

Control Wistar Rat Islets (A) compared with GK Rat Islets (B). Islets from GK and Wistar rats were incubated in the presence of glucose 2.8 mM or 16.7 mM. Insulin levels were measured after 20 min of incubation. ***p<0.001 vs. respective control value; mean ± SEM; n = 6 wells with 6–10 islets per well. Imeglimin (but not Metformin) Amplifies Insulin Secretion from GK Rat Islets: Islets from GK rats were incubated in the presence of high (16.7 mM) glucose (grey bars) or with high glucose plus the indicated concentrations of imeglimin (C; open bars), metformin (D; yellow bars), or GLP1 as a control (blue bars; panels C and D). Significant increases in mean (± SEM) glucose-stimulated insulin release are noted vs. respective control values; *p<0.05, **p<0.01, ***p<0.001; n = 15 to 16 observations per group. Effects of imeglimin on Kinetics of Insulin Secretion from GK Rat Islets (E). Islets from GK rats were alternately perifused with 2.8 mM glucose for 10 minutes and 16.7 mM glucose with (red curve) or without (black curve) imeglimin (100μM) for 10 minutes (10 to 20 min) followed by perifusion with 2.8 mM for an additional 10 minutes. The insulin levels in the perifusate was measured every minute from 0 min to 30 min. Mean ± SEM insulin levels are shown (data are derived from 4 independent experiments for each group at each time point).

Fig 3. Imeglimin does not increase cAMP generation in isolated GK rat islets.

In the presence of high glucose and the phosphodiesterase inhibitor IBMX, GLP1 (0.1μM) treatment increased the cAMP content of GK islets (+95%, ***p<0.001; n = 9). However, imeglimin (100 μM), produced no effect to increase cAMP under the same conditions. Mean ± SEM values are shown (n = 10). An additional independent experiment was also performed; levels of cAMP in each tested condition were not different between the two experiments.

Fig 4. Imeglimin increases the NAD⁺ pool through increased synthesis.

Gallotannin Effect on NAD⁺ (A). Islets from GK rats were incubated in the presence of 16.7 mM glucose with or without imeglimin (100 μM), or nicotinamide (15 mM); compounds were administered alone or in combination with gallotannin (10 μM). NAD⁺ was measured after 20 min incubation; mean (n = 10 in each group) ± SEM values are shown; *p<0.05, ***p<0.001 vs. Control; ## p<0.01 vs. nicotinamide alone. iNAMPT Activity (B). Islets from GK rats were incubated in the presence of 16.7 mM glucose with or without Imeglimin (100 μM), or nicotinamide (2 mM or 15 mM), or the combination of imeglimin and 2 mM nicotinamide. Intracellular (i) NAMPT activity was then measured; mean ± SEM (n = 5–6 per group) values are shown. *p<0.05, **p<0.01 vs. Control. In an independent experiment, iNAMPT activity was induced by the combination of imeglimin (100 μM) and 1 mM nicotinamide (+42%; p<0.05 vs. both control and nicotinamide alone). NAMPT mRNA Levels (C). Results from two separate experiments (Right and Left panels) are shown. NAMPT gene expression was determined by RT-PCR in islets from GK rats that were incubated for 30 min in the presence of 2.8 mM glucose (hatched bar), 16.7 mM glucose (solid bars) or 16.7 mM glucose plus imeglimin (100 μM; open bars). Mean (± SEM; n = 9–10 observations per group) levels of NAMPT mRNA are shown as fold vs. 16.7 mM glucose alone; #p<0.05 vs. 2.8 mM glucose; *p<0.05; ***p<0.001 vs. 16.7 mM glucose.

Fig 5. Imeglimin effect on insulin secretion is resistant to diazoxide.

(A) Islets from GK rats were incubated in low (2.8 mM) glucose with or without diazoxide (400 μM), tolbutamide (500 μM), or a combination of both diazoxide and tolbutamide. (B) GK rat islets were incubated in high (16.7 mM) glucose with or without diazoxide (400 μM), imeglimin (100 μM), or a combination of both diazoxide and imeglimin. Samples were obtained after 20 min and subsequently assayed to determine insulin concentrations; *p<0.05, **p<0.01, vs. respective control value. Mean ± SEM values are shown.

Fig 6. Potential role of CD38 and NAD⁺ metabolites to enhance insulin secretion via increasing intracellular Ca²⁺ in response to glucose.

Measurement of Intracellular Ca²⁺ in Perifused GK Rat Islets (A). Islets from GK rats were perifused alternately with glucose 2.8 mM and 16.7 mM glucose without treatment for Controls (black curve), with imeglimin 100 μM (red curve) or with GLP1 0.1 μM (green curve) followed by a third period of perifusion with 2.8 mM glucose alone. Intracellular Ca²⁺ levels were measured from individual islets by successive excitation at 340 nm and 380 nm and detection of fluorescence emitted at 510 nm every 10 seconds. Results for each of the three groups (control, imeglimin, GLP1) are derived from 8 experiments with a total of 8 to 10 rats per group (8 rats for control and GLP1 groups, 10 for the imeglimin group). Insulin Secretion Response to Imeglimin and GLP1 With and Without CD38 Knockdown: Scrambled sequence siRNA control (SC-Control, solid bars) and CD38 siRNA (open bars) transfected GK rat islets were incubated for 20 min in high (16.7 mM) glucose with or without 100 μM imeglimin (B) or 0.1 μM GLP (C). Mean ± SEM (n = 15–20 per group) insulin release values are shown; *p<0.05 vs. respective control.

Fig 7. Proposed model for mechanism of imeglimin action in islet β-cells.

The effects of imeglimin in the context of glucose stimulation are highlighted in red (text and arrows).