Activation of Melatonin Signaling Promotes β-Cell Survival and Function

Abstract

Type 2 diabetes mellitus (T2DM) is characterized by pancreatic islet failure due to loss of β-cell secretory function and mass. Studies have identified a link between a variance in the gene encoding melatonin (MT) receptor 2, T2DM, and impaired insulin secretion. This genetic linkage raises the question whether MT signaling plays a role in regulation of β-cell function and survival in T2DM. To address this postulate, we used INS 832/13 cells to test whether activation of MT signaling attenuates proteotoxicity-induced β-cell apoptosis and through which molecular mechanism. We also used nondiabetic and T2DM human islets to test the potential of MT signaling to attenuate deleterious effects of glucotoxicity and T2DM on β-cell function. MT signaling in β-cells (with duration designed to mimic typical nightly exposure) significantly enhanced activation of the cAMP-dependent signal transduction pathway and attenuated proteotoxicity-induced β-cell apoptosis evidenced by reduced caspase-3 cleavage (∼40%), decreased activation of stress-activated protein kinase/Jun-amino-terminal kinase (∼50%) and diminished oxidative stress response. Activation of MT signaling in human islets was shown to restore glucose-stimulated insulin secretion in islets exposed to chronic hyperglycemia as well as in T2DM islets. Our data suggest that β-cell MT signaling is important for the regulation of β-cell survival and function and implies a preventative and therapeutic potential for preservation of β-cell mass and function in T2DM.

Figure 1.

MT receptor expression in β-cells. Representative examples of nondiabetic human (A) and rat (B) islets stained for insulin (green), MT-2 (red) and nuclei (blue). C and D, INS 832/13 cells stained for insulin (green), MT-2 (red), and nuclei (blue) treated with either MT (10 nM) for 1 hour or vehicle solution.

Figure 2.

Persistent activation of β-cell MT receptor signaling enhances cAMP-PKA-CREB signaling pathway. A, Graphical illustration of the experimental work flow. B–D, Phospho-CREB (serine 133), phospho-ERK1/2, and GAPDH (loading control) protein levels and quantification in INS 832/13 cells exposed for 10 minutes to basal (2.8 mM glucose), GLP-1 (10 nM) at 16.7 mM glucose, or GIP (100 nM) at 16.7 mM glucose stimulation after overnight culture (12–14 hours) in media containing either melatonin (1–100 nM) or vehicle (DMSO). DMSO vehicle treatment was administered to all non-MT conditions to correct for potential confounding effects of DMSO, which was used to prepare MT solutions. Data are expressed as mean ± SEM and an average of four to six independent experiments. *, P < .05 vs vehicle.

Figure 3.

Persistent activation of β-cell MT receptor signaling protects against β-cell proteotoxicity. A, Graphical illustration of the experimental work flow. B and C, Cleaved caspase-3, phospho-JNK, and GAPDH (loading control) protein levels in control INS 832/13 cells and in cells transduced at 400 MOI with h-IAPP adenovirus for 48 hours and exposed for the final 12–14 hours to media containing either MT (10 nM) or vehicle (DMSO). The graph represents the quantification of the cleaved form of caspase-3 and JNK phosphorylation at threonine 183 and tyrosine 185 (n = 3–5 independent experiments). Data are expressed as mean ± SEM. *, P < .05. D, Immunoblot detection of carbonyl groups introduced into proteins due to oxidative stress and derivatized to DNP in INS 832/13 cells transduced at 400 MOI with h-IAPP adenovirus for 48 hours and exposed for the final 12–14 hours to media containing either MT (10 nM) or vehicle (DMSO) (a representative example of five independent experiments is shown). DMSO vehicle treatment was administered to all non-MT conditions to correct for potential confounding effects of DMSO, which was used to prepare melatonin solutions.

Figure 4.

Persistent activation of β-cell MT receptor signaling modulates apoptotic and oxidative stress gene expression in response to β-cell proteotoxicity. Apoptosis RT² Profiler PCR (A) and oxidative stress RT² Profiler PCR (B) arrays demonstrating relative mRNA expression of 146 genes purported to mediate cellular apoptotic and oxidative stress pathways in INS 832/13 cells transduced with h-IAPP adenovirus for 48 hours and exposed to either MT (10 nM) or vehicle (DMSO). The graph represents mRNA levels expressed as fold change for MT (10 nM) vs vehicle (DMSO)-treated cells (n = 3 independent experiments for each array). Data are expressed as mean ± SEM. Dashed red line represents mRNA expression in vehicle (DMSO)-treated cells. Genes demonstrating notably significant changes vs vehicle are highlighted in red. DMSO vehicle treatment was administered to all non-MT conditions to correct for potential confounding effects of DMSO, which was used to prepare MT solutions.

Figure 5.

Persistent activation of β-cell MT receptor signaling improves β-cell dysfunction associated with glucotoxicity in human islets. A, Graphical illustration of the experimental work flow. B, Immunoblot detection of carbonyl groups introduced into proteins due to oxidative stress and derivatized to DNP in human islets previously incubated for 72 hours in standard RPMI 1640 media supplemented with either 5 mM glucose (control) or 25 mM glucose (glucotoxic conditions) and exposed for the final 12–14 hours to media containing MT (10 or 100 nM), MT receptor agonist (ramelteon, 1 nM), or vehicle (DMSO). C and D, Assessment of glucose and incretin (GLP-1, 10 nM)-stimulated insulin secretion in human islets previously incubated for 72 hours in standard RPMI 1640 media supplemented with either 5 mM glucose (control) or 25 mM glucose (glucotoxic conditions) and exposed for the final 12–14 hours to media containing MT (100 nM), MT receptor agonist (ramelteon, 1 nM), or vehicle (DMSO). DMSO vehicle treatment was administered to all non-MT/ramelteon conditions to correct for potential confounding effects of DMSO, which was used to prepare MT/ramelteon solutions. Data are expressed as mean ± SEM and an average of five independent islet shipments. *, P < .05 vs basal.

Figure 6.

Persistent activation of β-cell MT receptor signaling improves β-cell dysfunction in T2DM human islets. A, Graphical illustration of the experimental work flow. B and C, Assessment of glucose and incretin (GLP-1, 10 nM)-stimulated insulin secretion in T2DM human islets incubated in standard RPMI 1640 media and exposed to overnight incubation (12–14 hours) in media containing MT (100 nM), MT receptor agonist (ramelteon, 1 nM), or vehicle (DMSO). Data are expressed as mean ± SEM and an average of four independent islet shipments. *, P < .05 vs basal. DMSO vehicle treatment was administered to all non-MT/ramelteon conditions to correct for potential confounding effects of DMSO, which was used to prepare MT/ramelteon solutions.