Melatonin influences insulin secretion primarily via MT(1) receptors in rat insulinoma cells (INS-1) and mouse pancreatic islets

Several studies have revealed that melatonin affects the insulin secretion via MT(1) and MT(2) receptor isoforms. Owing to the lack of selective MT(1) receptor antagonists, we used RNA interference technology to generate an MT(1) knockdown in a clonal β-cell line to evaluate whether melatonin modulates insulin secretion specifically via the MT(1) receptor. Incubation experiments were carried out, and the insulin concentration in supernatants was measured using a radioimmunoassay. Furthermore, the intracellular cAMP was determined using an enzyme-linked immunosorbent assay. Real-time RT-PCR indicated that MT(1) knockdown resulted in a significant increase in the rIns1 mRNA and a significantly elevated basal insulin secretion of INS-1 cells. Incubation with melatonin decreased the amount of glucagon-like peptide 1 or inhibited the glucagon-stimulated insulin release of INS-1 cells, while, in MT(1) -knockdown cells, no melatonin-induced reduction in insulin secretion could be found. No decrease in 3-isobutyl-1-methylxanthine-stimulated intracellular cAMP in rMT(1) -knockdown cells was detectable after treatment with melatonin either, and immunocytochemistry proved that MT(1) knockdown abolished phosphorylation of cAMP-response-element-binding protein. In contrast to the INS-1 cells, preincubation with melatonin did not sensitize the insulin secretion of rMT(1) -knockdown cells. We also monitored insulin secretion from isolated islets of wild-type and melatonin-receptor knockout mice ex vivo. In islets of wild-type mice, melatonin treatment resulted in a decrease in insulin release, whereas melatonin treatment of islets from MT(1) knockout and MT(1/2) double-knockout mice did not show a significant effect. The data indicate that melatonin inhibits insulin secretion, primarily via the MT(1) receptor in rat INS-1 cells and isolated mouse islets.     

Evidence for a circadian rhythm of insulin release from perifused rat pancreatic islets

Summary This study aims to analyse a circadian rhythm of insulin secretion from isolated rat pancreatic islets in vitro and its potential modulation by melatonin, the concentrations of which change in vivo inversely to that of insulin. The circadian rhythm was evaluated in a perifusion system, adapted to the specific conditions of pancreatic islets. To determine rhythmicity of insulin secretion, 30-min fractions were collected continuously for investigative periods of 44 to 112 h. Insulin secretion in 10 experiments was analysed by using the MacAnova-program for period length (τ), the χ²-periodogram for test of significance (p < 0.001), and additionally the empirical cosine adaptation for amplitude and goodness-of-fit. Thereby a circadian pattern was observed with periods (τ) between 21.8 and 26.2 h. The period duration (mean ± SEM) was 23.59 ± 0.503 h, the overall mean insulin release 1038 ± 13 pmol/l and the mean amplitude 88 ± 17 pmol/l. Adding melatonin (10 nmol/l, t = 2 h) as a hormonal Zeitgeber during analysis of circadian insulin secretion phase-response studies show phase-shifts with approximately 9 h phase advance. Thereafter the circadian period was maintained, while the amplitude was enhanced. From this it is concluded that an endogenous circadian oscillator is located within the pancreatic islets of the rat that regulates circadian insulin secretion of the insulin-producing beta cells. The pacemaker is remarkably stable, because its periodicity is not affected by factors altering insulin secretion. In agreement with inhibitory influences of melatonin (range 0.5 nmol/l to 5 μmol/l) on the insulin response in vitro, the phase-responses support the contention that pancreatic beta cells may be targets for melatonin action. [Diabetologia (1998) 42: 1085–1092] Received: 11 December 1997 and in final revised form: 20 March 1998     

New evidence for a role of melatonin in glucose regulation

Glucose triggers insulin secretion of the pancreatic β-cells. The pineal hormone melatonin interferes in this process by inhibiting secretion and transmitting circadian timing information to the islets. Circadian insulin secretion is adapted to day/night changes through melatonin-dependent synchronization. In rats and mice, melatonin levels are high during the dark period, which is their active feeding period, while, in humans, melatonin levels are high during the overnight fasting and sleeping period. This implies a different read-out of melatonin signaling in day-active species, including man. Dysregulation of circadian secretion may be a key to the increase of type 2 diabetes (T2D). This review discusses the impact of melatonin on insulin secretion transmitted through both the pertussis-toxin-sensitive membrane receptors MT1 (MTNR1a) and MT2 (MTNR1b) and the second messengers cAMP, cGMP and IP3. This is an important topic since, in several genetic association studies, single nucleotide polymorphisms of the human MT2-receptor have been described as being causally linked with an elevated risk of developing T2D. This article summarizes interrelationships between melatonin and insulin in type 1 diabetic (T1D) and type 2 diabetic (T2D) rats and humans.     

Melatonin inhibits insulin secretion and decreases PKA levels without interfering with glucose metabolism in rat pancreatic islets

The effect of melatonin (0.1 microM) on freshly isolated islets from adult rats was investigated. Melatonin caused a marked decrease of insulin secretion by islets in response to glucose. The mechanism involved was then examined. Melatonin did not interfere with glucose metabolism as indicated by the measurement of glucose oxidation. However, the content of the protein kinase A (PKA) catalytic alpha-subunit was significantly decreased in islets exposed to melatonin for 1 hr in the presence of 8.3 mM glucose, whereas that of the protein kinase C (PKC) alpha-subunit remained unchanged. Melatonin also inhibited forskolin-induced insulin secretion, a well known activator of adenylate cyclase (AC) activity. This may explain the low content of insulin found in islets incubated in the presence of melatonin for 3 hr. In fact, 3',5' -cyclic adenosine monophosphate (cAMP), a product of AC activity, stimulates insulin synthesis. These findings led us to postulate that a down-regulation of the PKA signaling pathway may be the mechanism involved in the melatonin inhibition of the process of glucose-induced insulin secretion.     

Melatonin stimulates inositol-1,4,5-trisphosphate and Ca2+ release from INS1 insulinoma cells

The effects of melatonin in mammalian cells are exerted via specific receptors or are related to its free radical scavenging activity. It has previously been reported that melatonin inhibits insulin secretion in the pancreatic islets of the rat and in rat insulinoma INS1 cells via Gi-protein-coupled MT1 receptors and the cyclic adenosine 3',5'-monophosphate pathway. However, the inositol-1,4,5-trisphosphate (IP3) pathway is involved in the insulin secretory response as well, and the melatonin signal may play a part in its regulation. This paper addresses the involvement of the second messengers IP3 and intracellular Ca2+ ([Ca2+]i) in the signalling cascade of melatonin in the rat insulinoma INS1 cell, a model for the pancreatic beta-cell. For this purpose melatonin at concentrations ranging from 1 to 100 nmol/L, carbachol and the nonselective melatonin receptor antagonist luzindole were used to stimulate INS1 cell batches, followed by an IP3-mass assay and Ca2+ imaging. Molecular biological studies relating to the mRNA of IP3 receptor (IP3R) subtypes and their relative abundance in INS1 cells showed expression of IP3R-1, IP3R-2 and IP3R-3 mRNA. In conclusion, we found that in rat insulinoma INS1 cells there is a dose-dependent stimulation of IP3 release by melatonin, which is accompanied by a likewise transient increase in [Ca2+]i concentrations. The melatonin effect observed mimics carbachol action. It can be abolished by 30 micromol/L luzindole and is sustained in Ca2+-free medium, suggesting a mechanism that includes the depletion of Ca2+ from intracellular stores.     

Increased melatonin synthesis in pineal glands of rats in streptozotocin induced type 1 diabetes

It is well-documented that melatonin influences insulin secretion. The effects are mediated by specific, high-affinity, pertussis-toxin-sensitive, G protein-coupled membrane receptors (MT(1) as well MT(2)), which are present in both the pancreatic tissue and islets of rats and humans, as well as in rat insulinoma cells (INS1). Via the Gi-protein-adenylatecyclase-3',5'-cyclic adenosine monophosphate (cAMP) and, possibly, the guanylatecyclase-cGMP pathways, melatonin decreases insulin secretion, whereas, by activating the Gq-protein-phospholipase C-IP(3) pathway, it has the opposite effect. For further analysis of the interactions between melatonin and insulin, diabetic rats were investigated with respect to melatonin synthesis in the pineal gland and plasma insulin levels. In this context, recent investigations have proven that type 2 diabetic rats and humans display decreased melatonin levels, whereas type 1 diabetic IDDM rats or those with diabetes induced by streptozotocin (STZ) of the present study show increased plasma melatonin levels and elevated AA-NAT-mRNA. Furthermore, the mRNA of pineal insulin receptors and beta1-adrenoceptors, including the clock genes Per1 and Bmal1 and the clock-controlled output gene Dbp, increases in both young and middle-aged STZ rats. The results therefore indicate that the decreased insulin levels in STZ-induced type 1 diabetes are associated with higher melatonin plasma levels. In good agreement with earlier investigations, it was shown that the elevated insulin levels observed in type 2 diabetes, are associated with decreased melatonin levels. The results thus prove that a melatonin-insulin antagonism exists. Astonishingly, notwithstanding the drastic metabolic disturbances in STZ-diabetic rats, the diurnal rhythms of the parameters investigated are maintained.     

Activation of insulin and IGF-1 signaling pathways by melatonin through MT1 receptor in isolated rat pancreatic islets

Melatonin diminishes insulin release through the activation of MT1 receptors and a reduction in cAMP production in isolated pancreatic islets of neonate and adult rats and in INS-1 cells (an insulin-secreting cell line). The pancreas of pinealectomized rats exhibits degenerative pathological changes with low islet density, indicating that melatonin plays a role to ensure the functioning of pancreatic beta cells. By using immunoprecipitation and immunoblotting analysis we demonstrated, in isolated rat pancreatic islets, that melatonin induces insulin growth factor receptor (IGF-R) and insulin receptor (IR) tyrosine phosphorylation and mediates the activities of the PI3K/AKT and MEK/ERKs pathways, which are involved in cell survival and growth, respectively. Thus, the effects of melatonin on pancreatic islets do not involve a reduction in cAMP levels only. This indoleamine may regulate growth and differentiation of pancreatic islets by activating IGF-I and insulin receptor signaling pathways.     

Regulation of the expression of serotonin N-acetyltransferase gene in Japanese quail (Coturnix japonica): I. Rhythmic pattern and effect of light

Serotonin N-acetyltransferase [arylalkylamine N-acetyltransferase (AANAT); EC2.3.1.87] is the rate-limiting enzyme in melatonin synthesis, and its activity exhibits a diurnal rhythm similar to that of the melatonin content in the pineal gland and retina of Japanese quail. Studies were conducted to characterize the Japanese quail AANAT cDNA, and to evaluate the expression of AANAT mRNA in the pineal gland, the retina, and other peripheral tissues. The nucleic acid sequence of a 400 bp cDNA clone obtained by RT-PCR manifested 78 and 95% homology compared to the rat and chicken AANAT cDNA, respectively, while the deduced amino acid sequence homology was 82 and 99%, respectively. AANAT mRNA content in a single pineal gland or an aliquot of eye lysate was measured by a micro-lysate protection assay. The expression of AANAT mRNA in the pineal gland and the retina exhibited circadian rhythm with peak levels at night. AANAT mRNA was also detected in the testis, but did not display a rhythmic change over a 24 hr period. AANAT mRNA was not detected in other tissues studied. Darkness during the day did not increase the pineal AANAT mRNA levels. However, unexpected light-exposure for 2 hr just after lights-off blocked the increase in AANAT mRNA, and at midnight remarkably decreased AANAT mRNA by 50%.     

Daily rhythm of glucose-induced insulin secretion by isolated islets from intact and pinealectomized rat

It is well known that pinealectomy induces in rats a diminished glucose tolerance, insulin resistance, a reduction in GLUT4 content in adipose and muscular tissues, a decrease in hepatic and muscular glycogenesis, impairment of glucagon action and an increase in blood pyruvate concentration. In addition, it has been shown that melatonin suppresses insulin secretion in several experimental conditions. The objective of the present study was to investigate the daily rhythm of glucose-induced insulin secretion and glucose oxidation by isolated pancreatic islets and to investigate the effect of chronic absence of melatonin (30 days of pinealectomy) on this rhythmic process. The data obtained confirmed the presence of a strong 24-hr rhythm of insulin secretion by isolated pancreatic islets. In addition, it was demonstrated that the glucose-metabolizing ability of the B-cell follows a daily rhythm phase locked to insulin secretion rhythm. Most interesting, however, was the demonstration that the daily rhythmic processes of insulin secretion and B-cell -[U-14C]-glucose oxidation by isolated pancreatic islets is completely modified by the chronic absence of the pineal gland. Thus, pinealectomy induced in all groups an increase in 24-hr mean glucose-stimulated insulin secretion and [U-14C]-glucose oxidation, in addition to some alterations in the rhythmic amplitude and a remarkable phase-advancing of the daily curves for 8.3 mm glucose (a condition similar to that observed in fed animals and where the B-cells are supposedly more active). These observations strongly suggest that the presence of the pineal gland may be necessary for the proper synchronization of these metabolic rhythms with other circadian rhythms like activity-rest and feeding.     

Placental melatonin system is present throughout pregnancy and regulates villous trophoblast differentiation

Melatonin is highly produced in the placenta where it protects against molecular damage and cellular dysfunction arising from hypoxia/re‐oxygenation‐induced oxidative stress as observed in primary cultures of syncytiotrophoblast. However, little is known about melatonin and its receptors in the human placenta throughout pregnancy and their role in villous trophoblast development. The purpose of this study was to determine melatonin‐synthesizing enzymes, arylalkylamine N‐acetyltransferase (AANAT) and hydroxyindole O‐methyltransferase (HIOMT), and melatonin receptors (MT1 and MT2) expression throughout pregnancy as well as the role of melatonin and its receptors in villous trophoblast syncytialization. Our data show that the melatonin generating system is expressed throughout pregnancy (from week 7 to term) in placental tissues. AANAT and HIOMT show maximal expression at the 3rd trimester of pregnancy. MT1 receptor expression is maximal at the 1st trimester compared to the 2nd and 3rd trimesters, while MT2 receptor expression does not change significantly during pregnancy. Moreover, during primary villous cytotrophoblast syncytialization, MT1 receptor expression increases, while MT2 receptor expression decreases. Treatment of primary villous cytotrophoblast with an increasing concentration of melatonin (10 pm –1 mm ) increases the fusion index (syncytium formation; 21% augmentation at 1 mm melatonin vs. vehicle) and β‐hCG secretion (121% augmentation at 1 mm melatonin vs. vehicle). This effect of melatonin appears to be mediated via its MT1 and MT2 receptors. In sum, melatonin machinery (synthetizing enzymes and receptors) is expressed in human placenta throughout pregnancy and promotes syncytium formation, suggesting an essential role of this indolamine in placental function and pregnancy well‐being.     

Long‐term enteral administration of melatonin reduces plasma insulin and increases expression of pineal insulin receptors in both Wistar and type 2‐diabetic Goto‐Kakizaki rats

Abstract: This paper represents an essential aspect of recent investigations into the functional and clinical implications of insulin–melatonin interrelationships. The aim of the study was to analyze whether melatonin reduces insulin secretion in an animal in a manner comparable to the pattern observed in previous in vitro experiments; to this end, we used two models: Wistar and type 2‐diabetic Goto‐Kakizaki (GK) rats. Thirty‐two Wistar and 32 GK rats were divided into two subgroups of 16 rats each; each subgroup was treated either with or without melatonin. The daily administration of melatonin, starting in 8‐ wk‐old rats, was adjusted to 2.5 mg/kg body weight. Melatonin was given daily during the dark period for 12 hr. After 9 wk of treatment, the rats were sacrificed in the middle of the dark period. Melatonin administration strongly enhanced the plasma melatonin level and diminished the expression of pancreatic melatonin receptor‐mRNA, whereas the expression of pineal AA‐NAT and HIOMT was unchanged. Furthermore, the experiments showed in agreement with recent in vitro results of pancreatic islets that plasma insulin levels were diminished after melatonin treatment. However, the pineal insulin receptor expression was increased after melatonin administration. The pancreatic expression of glucagon, GLUT2, and glucokinase was decreased in GK rats, whereas the glucose levels, as well as the parameters of glucose sensing, GLUT2‐mRNA, and glucokinase‐mRNA, were unchanged after melatonin administration in both Wistar and GK rats. In summary, the results show that melatonin administration decreases plasma insulin levels in vivo and, furthermore, that an insulin–melatonin antagonism exists.     

Analysis of the daily changes of melatonin receptors in the rat liver

Melatonin membrane (MT1 and MT2) and nuclear (RORα) receptors have been identified in several mammalian tissues, including the liver. The mechanisms regulating hepatic melatonin receptors are yet unknown. This study investigated whether these receptors exhibit daily changes and the effects of melatonin on their levels. Our results show that mRNAs for MT1/MT2 receptors exhibit circadian rhythms that were followed by rhythms in their respective protein levels; the acrophases for the two rhythms were reached at 04:00 and 05:00 hr, respectively. Pinealectomy blunted the rhythms in both mRNAs and protein levels. In contrast, mRNA and protein levels of nuclear receptor RORα increased significantly after pinealectomy. The cycles of the latter receptor also exhibited circadian rhythms which peaked at 03:00 and 03:45 hr, respectively. Melatonin administration (10–200 mg/kg) increased in a dose‐dependent manner the protein content of MT1/MT2 receptors, with no effects on RORα. Lunzindole treatment, however, did not affect melatonin receptor expression or content of either the membrane or nuclear receptors. Together with previously published findings which demonstrated the intracellular distribution of melatonin in rat liver, the current results support the conclusion that the circadian rhythms of MT1/MT2 and RORα receptors are under the control of the serum and intracellular melatonin levels. Moreover, the induction of MT1/MT2 receptors after the administration of high doses of melatonin further suggests that the therapeutic value of melatonin may not be restricted to only low doses of the indoleamine.     

The melatonin receptor subtype MT1 is expressed in human gallbladder epithelia

Based on the fact that human bile and, particularly gallbladder bile, contains high physiological levels of the antioxidant melatonin, the aim of this study was to investigate whether the melatonin receptor MT1 is present in human gallbladder. Expression and localization of MT1 was assessed by RT-PCR, Western blotting and immunofluorescence analysis in gallbladder samples from patients with cholelithiasis and with advanced gallbladder carcinoma. Additionally, we monitored mRNA expression of the two key enzymes of melatonin synthesis, i.e. arylalkylamine-N-acetyltransferase (AANAT) and hydroxyindole-O-methyltransferase (HIOMT). MT1 mRNA and protein were present in all cholelithiasis (n = 10) and gallbladder carcinoma (n = 5) samples. As indicated from RT-PCR and Western blot studies, MT1 is located in gallbladder epithelia. Epithelial expression was further proven by immunofluorescence staining of MT1 in paraffin-embedded cholelithiasis and gallbladder carcinoma sections. Analysis of AANAT and HIOMT mRNA expression showed that HIOMT mRNA is present in gallbladder. Surprisingly, AANAT was not detectable under conditions where it was found in a human colon specimen. The absence of AANAT suggests that in human gallbladder, HIOMT might be involved in the formation of 5-hydroxytryptamine products other than melatonin. In summary, our results provide the first evidence for the presence of MT1 in human gallbladder epithelia. Therefore, in addition to its profound antioxidative effects in the biliary system, melatonin might also act through MT1-mediated signal transduction pathways. Thereby, it might be involved in the regulation of gallbladder function.     

Melatonin attenuates smoking‐induced hyperglycemia via preserving insulin secretion and hepatic glycogen synthesis in rats

Epidemiology survey indicated that cigarette smoking is a risk factor of diabetes. However, the precise mechanisms remain to be clarified. In this study, we found that smoking caused metabolic malfunctions on pancreas and liver in experimental animal model. These were indicated by hyperglycemia, increased serum hemoglobin A1c level and decreased insulin secretion, inhibition of liver glycogen synthase (LGS), and hepatic glycogen synthesis. Mechanistic studies revealed that all these alterations were caused by the inflammatory reaction and reactive oxygen species (ROS) induced by the smoking. Melatonin treatment significantly preserved the functions of both pancreas and liver by reducing β cell apoptosis, CD68‐cell infiltration, ROS production, and caspase‐3 expression. The siRNA‐knockdown model identified that the protective effects of melatonin were mediated by melatonin receptor‐2 (MT2). This study uncovered potentially underlying mechanisms related to the association between smoking and diabetes. In addition, it is, for first time, to report that melatonin effectively protects against smoking‐induced glucose metabolic alterations and the signal transduction pathway of melatonin is mainly mediated by its MT2 receptor. These observations provide solid evidence for the clinically use of melatonin to reduce smoking‐related diabetes, and the therapeutic regimens are absent currently.