Identification and functional characterization of melatonin Mel 1a receptors in pancreatic β cells: potential role in incretin-mediated cell function by sensitization of cAMP signaling

Abstract

Melatonin receptors are expressed within the pancreatic islets of Langerhans, and melatonin induces a direct effect on insulin secretion ex-vivo. Here, we report the endogenous expression of the melatonin Mel 1a receptor in the INS-1 pancreatic β cell line. Pharmacological characterization of the receptor using a CRE-luciferase reporter gene demonstrated its functional activity in INS-1 cells, displaying the characteristic signaling properties of the G_i/o coupled receptor. Acute melatonin treatment of INS-1 cells in the presence of either forskolin or the incretin hormone glucagon-like peptide 1 (GLP-1) caused an attenuation of the responses in insulin secretion, insulin promoter activity, and CRE mediated gene expression, consistent with its effects in inhibiting cAMP mediated signal transduction. However, prolonged exposure (12 h) of INS-1 cells to melatonin treatment resulted in a sensitization of cAMP mediated responses to forskolin and GLP-1. Insulin secretion, insulin promoter activity and CRE mediated gene expression levels were augmented compared with responses without melatonin pre-treatment in INS-1 cells. In isolated rat islets, insulin secretion was enhanced following melatonin pre-treatment both in the absence and presence of GLP-1 or forskolin. This phenomenon reflects observations reported in other cell types expressing the melatonin Mel 1a receptor, and may represent the first evidence of a specific physiological role for melatonin-induced sensitization.

Introduction

Melatonin is synthesized in the pineal gland under strict regulation by environmental light and dark cues. This is reflected by very low levels of circulating melatonin in the daytime and high levels at night (Borjigin et al., 1999, Wilkinson et al., 1977). In addition to mediating the direct effects of light, melatonin is also involved in the entrainment of circadian and seasonal rhythms (Cassone et al., 1986, Redman et al., 1983). Although endogenous circadian rhythms are present in many tissues of the body, melatonin is necessary to synchronize the free-running rhythms and to dictate the phase relative to night and day (Redman et al., 1983).

Analysis of several reports in the literature suggest a possible inter-dependency between the function of the pineal gland and glucose homeostasis with possible significance with respect to the aetiology of diabetes. In one study, pinealectomized rats displayed elevated levels of blood glucose compared with control animals, while insulin concentrations were significantly lower. These effects were significantly reversed by melatonin treatment. Furthermore, the pinealectomized rats presented a marked glucose intolerance, which was also significantly attenuated by treatment with melatonin (Diaz and Blazquez, 1986). This effect on insulin secretion possibly suggests a direct role of melatonin on pancreatic function. In aged patients, a deterioration of melatonin circadian periodicity has been correlated with the occurrence of diabetes (Murialdo et al., 1993) and moreover, the circadian rhythm of melatonin was absent in diabetic autonomic neuropathy patients (O'Brien et al., 1986). Thus, in contrast to the findings in the pinealectomized rat model, these studies suggest that dysregulation of melatonin secretion may occur as a result of the diabetic state. This is further supported by evidence that experimentally induced diabetes reduces nocturnal pineal melatonin content in the Syrian hamster (Champney et al., 1983). It may be hypothesized therefore that a degree of symbiosis exists between the function of the pineal gland and the pancreas. In this study, we have addressed the question of whether melatonin imparts a direct functional effect on β cells of the islets of Langerhans.

Cellular effects of melatonin are mediated by specific high-affinity melatonin receptors, and available data indicate that the receptors are membrane associated and coupled to GTP-binding proteins (Morgan et al., 1989, Rivkees et al., 1989). Of the three melatonin receptor subtypes currently identified, only two (Mel 1a and Mel 1b) have been found in higher vertebrates and humans (Reppert et al., 1995a, Reppert et al., 1995b, Reppert et al., 1994). In cells expressing the receptors, melatonin inhibits forskolin-induced increase of cAMP accumulation (Browning et al., 2000). Pre-treatment with pertussis toxin abolishes the inhibitory effect, indicating that the receptor acts via G_i/G_o proteins (Barrett et al., 1999, Garcia-Perganeda et al., 1999).

The distribution of melatonin receptors has so far been primarily identified in specific areas of the brain [reviewed in Vanecek (1998)], with some data suggesting the localization of the melatonin receptor in peripheral sites including pituitary gonadotrophs and dermal melanophores (Ebisawa et al., 1994, Martin et al., 1982). Here, we examined the possibility of a further peripheral site of action of melatonin, the β cells of the pancreas.

Evidence that insulin secretion from pancreatic β cells is regulated in a circadian rhythm was established by perifusion analysis of freshly isolated rat pancreatic islets (Delattre et al., 1999, Peschke and Peschke, 1998) and also by analysis of plasma insulin levels in humans (Boden et al., 1996). The islets retained their endogenous rhythmicity several days following extraction from the host animal. In addition to this evidence of an endogenous circadian rhythm in islets, the investigators showed that the addition of melatonin to the medium reset the phase, suggesting that a mechanism of melatonin-induced entrainment of rhythmic phase may indeed be applicable to pancreatic function. Further support for melatonin action in islets was recently provided by the identification of high affinity binding sites for melatonin in isolated pancreatic islets of neonatal rats (Peschke et al., 2000). These reports provide suggestive evidence that melatonin receptors are present on β cells, although direct evidence of this has not yet been reported. Here, we have identified the presence of endogenous melatonin receptors in INS-1 β cells. Furthermore, we provide evidence that the rhythmic production of melatonin in-vivo may play a direct fascilitatory role in β cell function by sensitizing the cells to signaling by the incretin hormone glucagon-like peptide 1 (GLP-1).