Melatonin prevents deregulation of the sphingosine kinase/sphingosine 1‐phosphate signaling pathway in a mouse model of diethylnitrosamine‐induced hepatocellular carcinoma

The sphingosine kinase (SphK)/sphingosine 1‐phosphate (S1P) pathway is involved in multiple biological processes, including carcinogenesis. Melatonin shows beneficial effects in cell and animal models of hepatocellular carcinoma, but it is unknown if they are associated with the modulation of the SphK/S1P system, along with different downstream signaling pathways modified in cancer. We investigated the effects of melatonin in mice which received diethylnitrosamine (DEN) (35 mg/kg body weight i.p) once a week for 8 weeks. Melatonin was given at 5 or 10 mg/kg/day i.p. beginning 4 weeks after the onset of DEN administration and ending at the sacrifice time (10, 20, 30, or 40 weeks). Melatonin alleviated the distortion of normal hepatic architecture, lowered the incidence of preneoplastic/neoplastic lesions, and inhibited the expression of proliferative/cell cycle regulatory proteins (Ki67, PCNA, cyclin D1, cyclin E, CDK4, and CDK6). S1P levels and expression of SphK1, SphK2, and S1P receptors (S1PR1/S1PR3) were significantly elevated in DEN‐treated mice. However, there was a decreased expression of S1P lyase. These effects were significantly abrogated in a time‐ and dose‐dependent manner by melatonin, which also increased S1PR2 expression. Following DEN treatment, mice exhibited increased phosphorylation of PI3K, AKT, mTOR, STAT3, ERK, and p38, and a higher expression of NF‐κB p50 and p65 subunits. Melatonin administration significantly inhibited those changes. Data obtained suggest a contribution of the SphK/S1P system and related signaling pathways to the protective effects of melatonin in hepatocarcinogenesis.     

Melatonin modulates the autophagic response in acute liver failure induced by the rabbit hemorrhagic disease virus

Autophagy is an important survival pathway and participates in the host response to infection. Beneficial effects of melatonin have been previously reported in an animal model of acute liver failure (ALF) induced by the rabbit hemorrhagic disease virus (RHDV). This study was aimed to investigate whether melatonin protection against liver injury induced by the RHDV associates to modulation of autophagy. Rabbits were infected with 2 × 10⁴ hemagglutination units of a RHDV isolate and received 20 mg/kg melatonin at 0, 12, and 24 hr postinfection. RHDV induced autophagy, with increased expression of beclin‐1, ubiquitin‐like autophagy‐related (Atg)5, Atg12, Atg16L1 and sequestrosome 1 (p62/SQSTM1), protein 1 light chain 3 (LC3) staining, and conversion of LC3‐I to autophagosome‐associated LC3‐II. These effects reached a maximum at 24 hr postinfection, in parallel to extensive colocalization of LC3 and lysosome‐associated membrane protein (LAMP)‐1. The autophagic response induced by RHDV infection was significantly inhibited by melatonin administration. Melatonin treatment also resulted in decreased immunoreactivity for RHDV viral VP60 antigen and a significantly reduction in RHDV VP60 mRNA levels, oxidized to reduced glutathione ratio (GSSG/GSH), caspase‐3 activity, and immunoglobulin‐heavy‐chain‐binding protein (BiP) and CCAAT/enhancer‐binding protein homologous protein (CHOP) expression. Results indicate that, in addition to its antioxidant and antiapoptotic effects, and the suppression of ER stress, melatonin induces a decrease in autophagy associated with RHDV infection and inhibits RHDV RNA replication. Results obtained reveal novel molecular pathways accounting for the protective effect of melatonin in this animal model of ALF.     

Protective effect of melatonin against Opisthorchis viverrini‐induced oxidative and nitrosative DNA damage and liver injury in hamsters

Abstract: The liver fluke, Opisthorchis viverrini, is the risk factor of cholangiocarcinoma, which is a major health problem in northeastern Thailand. Production of reactive oxygen and nitrogen species during the host’s response leads to oxidative and nitrosative stress contributing to carcinogenesis. We investigated the protective effect of melatonin against O. viverrini‐induced oxidative and nitrosative stress and liver injury. Hamsters were infected with O. viverrini followed by oral administration of various doses of melatonin (5, 10, and 20 mg/kg body weight) for 30 days. Uninfected hamsters served as controls. Compared to the levels in O. viverrini‐infected hamsters without melatonin treatment, the indoleamine decreased the formation of oxidative and nitrosative DNA lesions, 8‐oxo‐7,8‐dihydro‐2′‐deoxyguanosine and 8‐nitroguanine, in the nucleus of bile duct epithelium and inflammatory cells, in parallel with a reduction in 3‐nitrotyrosine. Melatonin also reduced the expression of heme oxygenase‐1 and cytokeratin 19, nitrate/nitrite levels, and bile duct proliferation in the liver. Alanine transaminase activity and the levels of 8‐isoprostane and vitamin E were also dose dependently decreased in the plasma of melatonin‐treated hamsters. Melatonin reduced the mRNA expression of oxidant‐generating genes [inducible nitric oxide synthase, nuclear factor‐kappa B (NF‐κB), and cyclooxygenase‐2] and proinflammatory cytokines (TNF‐α and IL‐1β), accompanied by an increase in the expression of antioxidant genes [nuclear erythroid 2‐related factor 2 (Nrf2) and manganese superoxide dismutase]. Thus, melatonin may be an effective chemopreventive agent against O. viverrini‐induced cholangiocarcinoma by reducing oxidative and nitrosative DNA damage via induction of Nrf2 and inhibition of NF‐κB‐mediated pathways.     

Melatonergic system‐based two‐gene index is prognostic in human gliomas

Gliomas, the most common primary brain tumors in adults, are classified into four malignancy grades according to morphological features. Recent studies have shown that melatonin treatment induces cytotoxicity in glioma‐initiating cells and reduces the invasion and migration of glioma cell lines, inhibiting the nuclear factor κB (NFκB) oncopathway. Given that C6 rat glioma cells produce melatonin, we investigated the correlation between the capacity of gliomas to synthesize/metabolize melatonin and their overall malignancy. We first characterized the melatonergic system of human gliomas cell lines with different grades of aggressiveness (HOG, T98G, and U87MG) and demonstrated that glioma‐synthesized melatonin exerts an autocrine antiproliferative effect. Accordingly, the sensitivity to exogenous melatonin was higher for the most aggressive cell line, U87MG, which synthesized/accumulated less melatonin. Using The Cancer Genome Atlas RNAseq data of 351 glioma patients, we designed a predictive model of the content of melatonin in the tumor microenvironment, the ASMT:CYP1B1 index, combining the gene expression levels of melatonin synthesis and metabolism enzymes. The ASMT:CYP1B1 index negatively correlated with tumor grade, as well as with the expression of pro‐proliferation and anti‐apoptotic NFκB target genes. More importantly, the index was a grade‐ and histological type‐independent prognostic factor. Even when considering only high‐grade glioma patients, a low ASMT:CYP1B1 value, which suggests decreased melatonin and enhanced aggressiveness, was strongly associated with poor survival. Overall, our data reveal the prognostic value of the melatonergic system of gliomas and provide insights into the therapeutic role of melatonin.     

In vivo1H magnetic resonance spectroscopy‐derived metabolite variations between acute‐on‐chronic liver failure and acute liver failure

Background and Aims: Acute‐on‐chronic liver failure (ACLF), acute liver failure (ALF) and chronic liver disease (CLD) are common forms of liver failure and present with similar clinical profiles. The aim of this study was to compare brain metabolite alterations in all the three groups of patients with controls, using in vivo proton magnetic resonance spectroscopy (MRS), and to look for any significant differences in metabolites that may help in differentiating between these three conditions. Methods: Nine patients with ACLF, 10 with ALF, 10 patients with CLD and 10 age‐matched controls were studied. The relative concentrations of N‐acetylaspartate (NAA), choline (Cho), glutamine/glutamate (Glx) and myoinositol (mI) with respect to creatine (Cr) were measured. Results: ACLF (3.07±0.72), ALF (4.39±1.25) and CLD (3.15±0.69) patients exhibited significantly increased Glx/Cr ratios compared with controls (2.14±0.42). The NAA/Cr ratio was significantly decreased in both ACLF (mean=0.84±0.28) and CLD (mean=0.97±0.21) patients as compared with that in controls (mean=1.24±0.20). No significant difference among ALF, ACLF and CLD patients was noted in the Cho/Cr ratios. ACLF patients showed significantly lower mI/Cr and Glx/Cr ratios compared with the ALF group. Conclusion: In vivo proton MRS‐derived cerebral metabolite alterations in hepatic encephalopathy owing to ALF are significantly different from the one owing to ACLF and CLD; these may be due to the differences in the pathogenesis of these two overlapping clinical conditions.     

Sphingosine kinase 1 dependent protein kinase C-δ activation plays an important role in acute liver failure in mice

AIM: To investigate the role of protein kinase C (PKC)-δ activation in the pathogenesis of acute liver failure (ALF) in a well-characterized mouse model of D-galactosamine (D-GalN)/lipopolysaccharide (LPS)-induced ALF.METHODS: BALB/c mice were randomly assigned to five groups, and ALF was induced in mice by intraperitoneal injection of D-GaIN (600 mg/kg) and LPS (10 μg/kg). Kaplan-Meier method was used for survival analysis. Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels at different time points within one week were determined using a multiparameteric analyzer. Serum levels of high-mobility group box 1 (HMGB1), tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6, and IL-10 as well as nuclear factor (NF)-κB activity were determined by enzyme-linked immunosorbent assay. Hepatic morphological changes at 36 h after ALF induction were assessed by hematoxylin and eosin staining. Expression of PKC-δ in liver tissue and peripheral blood mononuclear cells (PBMCs) was analyzed by Western blot.RESULTS: The expression and activation of PKC-δ were up-regulated in liver tissue and PBMCs of mice with D-GalN/LPS-induced ALF. Inhibition of PKC-δ activation with rottlerin significantly increased the survival rates and decreased serum ALT/AST levels at 6, 12 and 24 h compared with the control group (P < 0.001). Rottlerin treatment also significantly decreased serum levels of HMGB1 at 6, 12, and 24 h, TNF-α, IL-6 and IL-1 β at 12 h compared with the control group (P < 0.01). The inflammatory cell infiltration and necrosis in liver tissue were also decreased in the rottlerin treatment group. Furthermore, sphingosine kinase 1 (SphK1) dependent PKC-δ activation played an important role in promoting NF-κB activation and inflammatory cytokine production in ALF.CONCLUSION: SphK1 dependent PKC-δ activation plays an important role in promoting NF-κB activation and inflammatory response in ALF, and inhibition of PKC-δ activation might be a potential therapeutic strategy for this disease.     

Melatonin promotes hepatic differentiation of human dental pulp stem cells: clinical implications for the prevention of liver fibrosis

Melatonin's effect on hepatic differentiation of stem cells remains unclear. The aim of this study was to investigate the action of melatonin on hepatic differentiation as well as its related signaling pathways of human dental pulp stem cells (hDPSCs) and to examine the therapeutic effects of a combination of melatonin and hDPSC transplantation on carbon tetrachloride (CCl₄)‐induced liver fibrosis in mice. In vitro hepatic differentiation was assessed by periodic acid‐Schiff (PAS) staining and mRNA expression for hepatocyte markers. Liver fibrosis model was established by injecting 0.5 mL/kg CCl₄ followed by treatment with melatonin (5 mg/kg, twice a week) and hDPSCs. In vivo therapeutic effects were evaluated by histopathology and by means of liver function tests including measurement of alanine transaminase (ALT), aspartate transaminase (AST), and ammonia levels. Melatonin promoted hepatic differentiation based on mRNA expression of differentiation markers and PAS‐stained glycogen‐laden cells. In addition, melatonin increased bone morphogenic protein (BMP)‐2 expression and Smad1/5/8 phosphorylation, which was blocked by the BMP antagonist noggin. Furthermore, melatonin activated p38, extracellular signal‐regulated kinase (ERK), and nuclear factor‐κB (NF‐κB) in hDPSCs. Melatonin‐induced hepatic differentiation was attenuated by inhibitors of BMP, p38, ERK, and NF‐κB. Compared to treatment of CCl₄‐injured mice with either melatonin or hDPSC transplantation alone, the combination of melatonin and hDPSC significantly suppressed liver fibrosis and restored ALT, AST, and ammonia levels. For the first time, this study demonstrates that melatonin promotes hepatic differentiation of hDPSCs by modulating the BMP, p38, ERK, and NF‐κB pathway. Combined treatment of grafted hDPSCs and melatonin could be a viable approach for the treatment of liver cirrhosis.     

Melatonin overcomes gemcitabine resistance in pancreatic ductal adenocarcinoma by abrogating nuclear factor‐κB activation

Constitutive activation and gemcitabine induction of nuclear factor‐κB (NF‐κB) contribute to the aggressive behavior and chemotherapeutic resistance of pancreatic ductal adenocarcinoma (PDAC). Thus, targeting the NF‐κB pathway has proven an insurmountable challenge for PDAC therapy. In this study, we investigated whether the inhibition of NF‐κB signaling pathway by melatonin might lead to tumor suppression and overcome gemcitabine resistance in pancreatic tumors. Our results showed that melatonin inhibited activities of NF‐κB by suppressing IκBα phosphorylation and decreased the expression of NF‐κB response genes in MiaPaCa‐2, AsPc‐1, Panc‐28 cells and gemcitabine resistance MiaPaCa‐2/GR cells. Moreover, melatonin not only inhibited cell proliferation and invasion in a receptor‐independent manner, but also enhanced gemcitabine cytotoxicity at pharmacologic concentrations in these PDAC cells. In vivo, the mice treated with both agents experienced a larger reduction in tumor burden than the single drug‐treated groups in an orthotopic xenograft mouse model. Taken together, these results indicate that melatonin inhibits proliferation and invasion of PDAC cells and overcomes gemcitabine resistance of pancreatic tumors through NF‐κB inhibition. Our findings therefore provide novel preclinical knowledge about melatonin inhibition of NF‐κB in PDAC and suggest that melatonin should be investigated clinically, alone or in combination with gemcitabine for PDAC treatment.     

Melatonin suppresses activation of hepatic stellate cells through RORα‐mediated inhibition of 5‐lipoxygenase

Liver fibrosis is scar tissue resulting from an uncontrolled wound‐healing process in response to chronic liver injury. Liver damage generates an inflammatory reaction that activates hepatic stellate cells (HSC) that transdifferentiate from quiescent cells that control retinol metabolism to proliferative and migratory myofibroblasts that produce excessive amounts of extracellular matrix proteins, in particular collagen 1a1 (COL1A1). Although liver fibrosis is reversible, no effective drug therapy is available to prevent or reverse HSC activation. Melatonin has potent hepatoprotective properties in a variety of acute and chronic liver injury models and suppresses liver fibrosis. However, it remains unclear whether melatonin acts indirectly or directly on HSC to prevent liver fibrosis. Here, we studied the effect of melatonin on culture‐activated rat HSC. Melatonin dose‐dependently suppressed the expression of HSC activation markers Col1a1 and alpha‐smooth muscle actin (αSMA, Acta2), as well as HSC proliferation and loss of lipid droplets. The nuclear melatonin sensor retinoic acid receptor‐related orphan receptor‐alpha (RORα/Nr1f1) was expressed in quiescent and activated HSC, while the membranous melatonin receptors (Mtrn1a and Mtrn1b) were not. The synthetic RORα agonist SR1078 more potently suppressed Col1a1 and αSma expression, HSC proliferation, and lipid droplet loss, while the RORα antagonist SR1001 blocked the antifibrotic features of melatonin. Melatonin and SR1078 inhibited the expression of Alox5, encoding 5‐lipoxygenase (5‐LO). The pharmacological 5‐LO inhibitor AA861 reduced Acta2 and Col1a1 expression in activated HSC. We conclude that melatonin directly suppresses HSC activation via RORα‐mediated inhibition of Alox5 expression, which provides novel drug targets to treat liver fibrosis.     

Analysis of N1‐acetyl‐N2‐formyl‐5‐methoxykynuramine/N1‐acetyl‐5‐methoxy‐kynuramine formation from melatonin in mice

Abstract: The interactions of melatonin, a potent endogenous antioxidant, with reactive oxygen species generate several products that include N¹‐acetyl‐N²‐formyl‐5‐methoxykynuramine (AFMK) and N¹‐acetyl‐5‐methoxy‐kynuramine (AMK). The physiological or pathological significance of AFMK/AMK formation during the process of melatonin metabolism in mammals has not been clarified. Using a metabolomic approach in the current study, the AFMK/AMK pathway was thoroughly investigated both in mice and humans. Unexpectedly, AFMK and AMK were not identified in the urine of humans nor in the urine, feces or tissues (including liver, brain, and eyes) in mice under the current experimental conditions. Metabolomic analysis did identify novel metabolites of AMK, i.e. hydroxy‐AMK and glucuronide‐conjugated hydroxy‐AMK. These two newly identified metabolites were, however, not found in the urine of humans. In addition, oxidative stress induced by acetaminophen in the mouse model did not boost AFMK/AMK formation. These data suggest that AFMK/AMK formation is not a significant pathway of melatonin disposition in mice, even under conditions of oxidative stress.     

Neuroprotective effect of melatonin against ischemia is partially mediated by alpha‐7 nicotinic receptor modulation and HO‐1 overexpression

Melatonin has been widely studied as a protective agent against oxidative stress. However, the molecular mechanisms underlying neuroprotection in neurodegeneration and ischemic stroke are not yet well understood. In this study, we evaluated the neuroprotective/antioxidant mechanism of action of melatonin in organotypic hippocampal cultures (OHCs) as well as in photothrombotic stroke model in vivo. Melatonin (0.1, 1, and 10 μm ) incubated postoxygen and glucose deprivation (OGD) showed a concentration‐dependent protection; maximum protection was achieved at 10 μm (90% protection). Next, OHCs were exposed to 10 μm melatonin at different post‐OGD times; the protective effect of melatonin was maintained at 0, 1, and 2 hr post‐OGD treatment, but it was lost at 6 hr post‐OGD. The protective effect of melatonin and the reduction in OGD‐induced ROS were prevented by luzindole (melatonin antagonist) and α‐bungarotoxin (α‐Bgt, a selective α7 nAChR antagonist). In Nrf2 knockout mice, the protective effect of melatonin was reduced by 40% compared with controls. Melatonin, incubated 0, 1, and 2 hr post‐OGD, increased the expression of heme oxygenase‐1 (HO‐1), and this overexpression was prevented by luzindole and α‐bungarotoxin. Finally, administration of 15 mg/kg melatonin following the induction of photothrombotic stroke in vivo, reduced infarct size (50%), and improved motor skills; this effect was partially lost in 0.1 mg/kg methyllycaconitine (MLA, selective α7 nAChR antagonist)‐treated mice. Taken together, these results demonstrate that postincubation of melatonin provides a protective effect that, at least in part, depends on nicotinic receptor activation and overexpression of HO‐1.     

Inhibition of sphingosine kinase 1 ameliorates acute liver failure by reducing high-mobility group box 1 cytoplasmic translocation in liver cells

AIM: To determine the therapeutic potential of sphingosine kinase 1(Sphk1) inhibition and its underlying mechanism in a well-characterized mouse model of D-galactosamine(D-Gal N)/lipopolysaccharide(LPS)-induced acute liver failure(ALF).METHODS: Balb/c mice were randomly assigned to different groups,with ALF induced by intraperitoneal injection of D-Ga IN(600 mg/kg) and LPS(10 μg/kg). The Kaplan-Meier method was used for survival analysis. Serum alanine aminotransferase(ALT) and aspartate aminotransferase(AST) levels at different time points within one week were determined using a multi-parametric analyzer. Serum high-mobility group box 1(HMGB1),tumor necrosis factor-α(TNF-α),interleukin(IL)-1β,IL-6,IL-10,and sphingosine-1-phosphate were detected by enzyme-linked immunosorbent assay. Hepatic morphological changes at 36 h after acute liver injury induction were assessed by hematoxylin and eosin staining. HMGB1 expression in hepatocytes and cytoplasmic translocation were detected by immunohistochemistry. Expression of Sphk1 in liver tissue and peripheral blood mononuclear cells(PBMCs) was analyzed by Western blot.RESULTS: The expression of Sphk1 in liver tissue and PBMCs was upregulated in Gal N/LPS-induced ALF. Upregulated Sphk1 expression in liver tissue was mainly caused by Kupffer cells,the resident macrophages of the liver. The survival rates of mice in the N,Ndimethylsphingosine(DMS,a specific inhibitor of Sph K1) treatment group were significantly higher than that of the control group(P 0.001). DMS treatment significantly decreased the levels of serum ALT and AST at 6,12,and 24 h compared with that of the control group(P 0.01 for all). Serum HMGB1 levels at 6,12,and 24 h,as well as serum TNF-α,IL-6,and IL-1β levels at 12 h,were significantly lower in the DMS treatment group than in the control group(P 0.01 for all). Furthermore,hepatic inflammation,necrosis,and HMGB1 cytoplasm translocation in liver cells were significantly decreased in the DMS treatment group compared to the control group(43.72% ± 5.51% vs 3.57% ± 0.83%,χ2 = 12.81,P 0.01).CONCLUSION: Inhibition of Sph K1 ameliorates ALF by reducing HMGB1 cytoplasmic translocation in liver cells,and so might be a potential therapeutic strategy for this disease.     

Melatonin prevents hemorrhagic shock‐induced liver injury in rats through an Akt‐dependent HO‐1 pathway

Abstract: Although melatonin treatment following trauma‐hemorrhage or ischemic reperfusion prevents organs from dysfunction and injury, the precise mechanism remains unknown. This study tested whether melatonin prevents liver injury following trauma‐hemorrhage involved the protein kinase B (Akt)‐dependent heme oxygenase (HO)‐1 pathway. After a 5‐cm midline laparotomy, male rats underwent hemorrhagic shock (mean blood pressure approximately 40 mmHg for 90 min) followed by fluid resuscitation. At the onset of resuscitation, rats were treated with vehicle, melatonin (2 mg/kg), or melatonin plus phosphoinositide 3‐kinase (PI3K) inhibitor wortmannin (1 mg/kg). At 2 hr after trauma‐hemorrhage, the liver tissue myeloperoxidase activity, malondialdehyde, adenosine triphosphate, serum alanine aminotransferase, and aspartate aminotransferase levels were significantly increased compared with sham‐operated control. Trauma‐hemorrhage resulted in a significant decrease in the Akt activation in comparison with the shams (relative density, 0.526 ± 0.031 versus 1.012 ± 0.066). Administration of melatonin following trauma‐hemorrhage normalized liver Akt phosphorylation (0.993 ± 0.061), further increased mammalian target of rapamycin (mTOR) activation (5.263 ± 0.338 versus 2.556 ± 0.225) and HO‐1 expression (5.285 ± 0.325 versus 2.546 ± 0.262), and reduced cleaved caspase‐3 levels (2.155 ± 0.297 versus 5.166 ± 0.309). Coadministration of wortmannin abolished the melatonin‐mediated attenuation of the shock‐induced liver injury markers. Our results collectively suggest that melatonin prevents hemorrhagic shock‐induced liver injury in rats through an Akt‐dependent HO‐1 pathway.     

Melatonin influences somatostatin secretion from human pancreatic δ‐cells via MT1 and MT2 receptors

Melatonin is an effector of the diurnal clock on pancreatic islets. The membrane receptor‐transmitted inhibitory influence of melatonin on insulin secretion is well established and contrasts with the reported stimulation of glucagon release from α‐cells. Virtually, nothing is known concerning the melatonin‐mediated effects on islet δ‐cells. Analysis of a human pancreatic δ‐cell model, the cell line QGP‐1, and the use of a somatostatin‐specific radioimmunoassay showed that melatonin primarily has an inhibitory effect on somatostatin secretion in the physiological concentration range. In the pharmacological range, melatonin elicited slightly increased somatostatin release from δ‐cells. Cyclic adenosine monophosphate (cAMP) is the major second messenger dose‐dependently stimulating somatostatin secretion, in experiments employing the membrane‐permeable 8‐Br‐cAMP. 8‐Br‐cyclic guanosine monophosphate proved to be of only minor relevance to somatostatin release. As the inhibitory effect of 1 nm melatonin was reversed after incubation of QGP‐1 cells with the nonselective melatonin receptor antagonist luzindole, but not with the MT2‐selective antagonist 4‐P‐PDOT (4‐phenyl‐2‐propionamidotetraline), an involvement of the MT1 receptor can be assumed. Somatostatin release from the δ‐cells at low glucose concentrations was significantly inhibited during co‐incubation with 1 nm melatonin, an effect which was less pronounced at higher glucose levels. Transient expression experiments, overexpressing MT1, MT2, or a deletion variant as a control, indicated that the MT1 and not the MT2 receptor was the major transmitter of the inhibitory melatonin effect. These data point to a significant influence of melatonin on pancreatic δ‐cells and on somatostatin release.