Distribution and density of melatonin receptors in human main pancreatic islet cell types.

Recent investigations of our group established that melatonin modulates hormone secretion of pancreatic islets via melatonin receptor types MT1 and MT2. Expression of MT1 and MT2 has been shown in mouse, rat, and human pancreatic islets as well as in the ß-, α-, and δ-cell lines INS-1, αTC1.9, and QGP-1. In view of these earlier investigations, this study was performed to analyze in detail the distribution and density of melatonin receptors on the main islet cell types in human pancreatic tissue obtained from nondiabetic and type 2 diabetic patients. Immunohistochemical analysis established the presence of MT1 and MT2 in ß-, α-, and δ-cells, but notably, with differences in receptor density. In general, the lowest MT1 and MT2 receptor density was measured in α-cells compared to the 2 other cell types. In type 2 diabetic islets, MT1 and MT2 receptor density was increased in δ-cells compared to normoglycemic controls. In human islets in batch culture of a nondiabetic donor, an increase of somatostatin secretion was observed under melatonin treatment while in islets of a type 2 diabetic donor, an inhibitory influence could be observed, especially in the presence of 5.5 mmol/L glucose. These data suggest the following: i) cell-type-specific density of MT1 and MT2 receptors in human pancreatic islets, which should be considered in context of the hormone secretion of islets, ii) the influence of diabetes on density of MT1 and MT2 as well as iii) the differential impact of melatonin on somatostatin secretion of nondiabetic and type 2 diabetic islets.

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.

Experimental and clinical aspects of melatonin and clock genes in diabetes.

The pineal hormone melatonin influences insulin secretion, as well as glucagon and somatostatin secretion, both in vivo and in vitro. These effects are mediated by two specific, high-affinity, seven transmembrane, pertussis toxin-sensitive, Gi-protein-coupled melatonin receptors, MT1 and MT2. Both isoforms are expressed in the ß-cells, α-cells as well as δ-cells of the pancreatic islets of Langerhans and are involved in the modulation of insulin secretion, leading to inhibition of the adenylate cyclase-dependent cyclic adenosine monophosphate as well as cyclic guanosine monophosphate formation in pancreatic ß-cells by inhibiting the soluble guanylate cyclase, probably via MT2 receptors. In this way, melatonin also likely inhibits insulin secretion, whereas using the inositol triphosphate pathway after previous blocking of Gi-proteins by pertussis toxin, melatonin increases insulin secretion. Desynchrony of receptor signaling may lead to the development of type 2 diabetes. This notion has recently been supported by genomewide association studies pinpointing variances of the MT2 receptor as a risk factor for this rapidly spreading metabolic disturbance. As melatonin is secreted in a clearly diurnal fashion, it is safe to assume that it also has a diurnal impact on the blood-glucose-regulating function of the islet. Observations of the circadian expression of clock genes (Clock, Bmal1, Per1,2,3, and Cry1,2) in pancreatic islets, as well as in INS1 rat insulinoma cells, may indicate that circadian rhythms are generated in the ß-cells themselves. The circadian secretion of insulin from pancreatic islets is clock-driven. Disruption of circadian rhythms and clock function leads to metabolic disturbances, for example, type 2 diabetes. The study of melatonin-insulin interactions in diabetic rat models has revealed an inverse relationship between these two hormones. Both type 2 diabetic rats and patients exhibit decreased melatonin levels and slightly increased insulin levels, whereas type 1 diabetic rats show extremely reduced levels or the absence of insulin, but statistically significant increases in melatonin levels. Briefly, an increase in melatonin levels leads to a decrease in stimulated insulin secretion and vice versa. Melatonin levels in blood plasma, as well as the activity of the key enzyme of melatonin synthesis, AA-NAT (arylalkylamine-N-acetyltransferase) in pineal, are lower in type 2 diabetic rats compared to controls. In contrast, melatonin and pineal AA-NAT mRNA are increased and insulin receptor mRNA is decreased in type 1 diabetic rats, which also indicates a close relationship between insulin and melatonin. As an explanation, it was hypothesized that catecholamines, which reduce insulin levels and stimulate melatonin synthesis, control insulin-melatonin interactions. This conviction stems from the observation that catecholamines are increased in type 1 but are diminished in type 2 diabetes. In this context, another important line of inquiry involves the fact that melatonin protects ß-cells against functional overcharge and, consequently, hinders the development of type 2 diabetes.

Effects of insulin on norepinephrine- and acetylcholine-induced membrane currents of pinealocytes from healthy Wistar and type 2 diabetic GK rats.

The neurohormone melatonin is synthesized by the pineal gland under the stimulation of norepinephrine (NE). Its synthesis is inhibited by acetylcholine (ACh) and by insulin. Type 2 diabetic Goto Kakizaki (GK) rats have higher insulin and lower melatonin plasma levels than healthy Wistar rats. We investigate membrane potentials and currents of isolated pinealocytes in both rat strains and the influence of NE, ACh and insulin by using the perforated patch whole cell clamp technique. Pinealocyte membranes displayed a high resting Na(+) conductance. Stimulation with NE further increased this Na(+) conductance, which led to a slight depolarization in unclamped cells. The amplitude of the NE-evoked current was similar in both rat strains but the current fraction carried by Na(+) was stronger in GK rats. Stimulation with ACh induced a transient inward current and depolarization. These effects were much more pronounced in the pinealocytes of GK rats. The NE-induced current, the ACh-induced current and the membrane depolarization were reduced by pre-administration of insulin in Wistar pinealocytes. Our results provide the first electrophysiological evidence for the modulation, by insulin, of the effects of NE and ACh in pinealocytes of normal rats. The pinealocytes of type 2 diabetic rats were not responsive to insulin. This might explain the reported correlation between the decreased insulin receptor mRNA transcript levels in GK rat pinealocytes and the lack of effect of insulin on ion channels in their cell membranes.

Calcium-signaling components in rat insulinoma ß-cells (INS-1) and pancreatic islets are differentially influenced by melatonin.

The pineal secretory product melatonin exerts its influence on the insulin secretion of pancreatic islets by different signaling pathways. The purpose of this study was to analyze the impact of melatonin on calcium-signaling components under different conditions. In a transfected INS-1 cell line overexpressing the human MT2 receptor (hMT2-INS-1), melatonin treatment induced even stronger depressive effects on calcium/calmodulin-dependent kinase 2d and IV (Camk2d, CamkIV) transcripts during 3-isobutyl-1-methylxanthine (IBMX) treatment than in normal INS-1 cells, indicating a crucial influence of melatonin receptor density on transcript-level regulation. In addition, melatonin induced a significant downregulation of calmodulin (Calm1) in IBMX-treated hMT2-INS-1 cells. Long-term administration of melatonin alone reduced CamkIV transcript levels in INS-1 cells; however, transcript levels of Camk2d remained unchanged. The release of insulin was diminished under long-term melatonin treatment. The impact of melatonin also involved reductions in CAMK2D protein during IBMX or forskolin treatments in INS-1 cells, as measured by an enzyme-linked immunosorbent assay, indicating a functional significance of transcriptional changes in pancreatic islets. Furthermore, analysis of melatonin receptor knockout mice showed that the transcript levels of Camk2d, CamkIV, and Calm1 were differentially influenced according to the melatonin receptor subtype deleted. In conclusion, this study provides evidence that melatonin has different impacts on the regulation of Calm1 and Camk. These calcium-signaling components are known as participants in the calcium/calmodulin pathway, which plays an important functional role in the modulation of the ß-cell signaling pathways leading to insulin secretion.

Influence of melatonin receptor signalling on parameters involved in blood glucose regulation.

The pineal hormone melatonin is known to influence insulin secretion via the G-protein-coupled receptor isoforms MT1 and MT2. The present study was aimed to further elucide the impact of melatonin on blood glucose regulation. To this end, mouse lines were used, in which one of the two or both melatonin receptors were deleted. In comparison with wild-type mice of the same age (8-12 months old), increased plasma insulin and melatonin levels and decreased blood glucose levels and body weights were detected in the MT1- and double-knockout lines. The elimination of melatonin receptor signalling also altered blood glucose concentrations, body weight and melatonin and insulin levels when comparing wild-type and receptor knockout mice of different ages (6 wk and 8-12 months old); such changes, however, were dependent on the type of receptor deleted. Furthermore, reverse transcription polymerase chain reaction results provided evidence that melatonin receptor deficiency has an impact on transcript levels of pancreatic islet hormones as well as on pancreatic and hepatic glucose transporters (Glut1 and 2). Under stimulated insulin secretion in the presence of melatonin in the rat insulinoma ß-cells INS-1, the Glut1 transcript level was decreased. In conclusion, the present findings demonstrate that melatonin receptor knockout types affect blood glucose levels, body weight, plasma levels of melatonin and insulin, as well as pancreatic hormone and Glut1 expression in significantly different manners.

Remodelling of the intracardiac ganglia in diabetic Goto-Kakizaki rats: an anatomical study.

BACKGROUND: Although cardiac autonomic neuropathy is one of major complications of diabetes mellitus (DM), anatomical data on cardiac innervation of diabetic animal models is scant and controversial. We performed this study to check whether long-term diabetic state impacts the anatomy of intracardiac ganglia in Goto-Kakizaki (GK) rats, a genetic model of type 2 DM. METHODS: Twelve GK rats (276 ± 17 days of age; mean ± standard error) and 13 metabolically healthy Wistar rats (262 ± 5 days of age) as controls were used for this study. Blood glucose was determined using test strips, plasma insulin by radioimmunoassay. Intrinsic ganglia and nerves were visualized by acetylcholinesterase histochemistry on whole hearts. Ganglion area was measured, and the neuronal number was assessed according to ganglion area. RESULTS: The GK rats had significantly elevated blood glucose level compared to controls (11.0 ± 0.6 vs. 5.9 ± 0.1 mmol/l, p < 0.001), but concentration of plasma insulin did not differ significantly between the two groups (84.0 ± 9.8 vs. 67.4 ± 10.9 pmol/l, p = 0.17). The GK rats contained significantly fewer intracardiac ganglia, decreased total area of intracardiac ganglia (1.4 ± 0.1 vs. 2.2 ± 0.1 mm2, p < 0.001) and smaller somata of ganglionic neurons. Mean total number of intracardiac neurons in GK rats was 1461 ± 62, while this number in control rats was higher by 39% and reached 2395 ± 110 (p < 0.001). CONCLUSIONS: Results of our study demonstrate the decreased number of intracardiac neurons in GK rats compared to metabolically healthy Wistar rats of similar age. It is likely that the observed structural remodelling of intracardiac ganglia in GK rats is caused by a long-term diabetic state.

Melatonin and pancreatic islets: interrelationships between melatonin, insulin and glucagon.

The pineal hormone melatonin exerts its influence in the periphery through activation of two specific trans-membrane receptors: MT1 and MT2. Both isoforms are expressed in the islet of Langerhans and are involved in the modulation of insulin secretion from ß-cells and in glucagon secretion from α-cells. De-synchrony of receptor signaling may lead to the development of type 2 diabetes. This notion has recently been supported by genome-wide association studies identifying particularly the MT2 as a risk factor for this rapidly spreading metabolic disturbance. Since melatonin is secreted in a clearly diurnal fashion, it is safe to assume that it also has a diurnal impact on the blood-glucose-regulating function of the islet. This factor has hitherto been underestimated; the disruption of diurnal signaling within the islet may be one of the most important mechanisms leading to metabolic disturbances. The study of melatonin-insulin interactions in diabetic rat models has revealed an inverse relationship: an increase in melatonin levels leads to a down-regulation of insulin secretion and vice versa. Elucidation of the possible inverse interrelationship in man may open new avenues in the therapy of diabetes.

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 ß-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 of the receptor-mediated influence of melatonin on pancreatic glucagon secretion via the Gαq protein-coupled and PI3K signaling pathways.

Melatonin has been shown to modulate glucose metabolism by influencing insulin secretion. Recent investigations have also indicated a regulatory function of melatonin on the pancreatic α-cells. The present in vitro and in vivo studies evaluated whether melatonin mediates its effects via melatonin receptors and which signaling cascade is involved. Incubation experiments using the glucagon-producing mouse pancreatic α-cell line αTC1 clone 9 (αTC1.9) as well as isolated pancreatic islets of rats and mice revealed that melatonin increases glucagon secretion. Preincubation of αTC1.9 cells with the melatonin receptor antagonists luzindole and 4P-PDOT abolished the glucagon-stimulatory effect of melatonin. In addition, glucagon secretion was lower in the pancreatic islets of melatonin receptor knockout mice than in the islets of the wild-type (WT) control animals. Investigations of melatonin receptor knockout mice revealed decreased plasma glucagon concentrations and elevated mRNA expression levels of the hepatic glucagon receptor when compared to WT mice. Furthermore, studies using pertussis toxin, as well as measurements of cAMP concentrations, ruled out the involvement of Gαi- and Gαs-coupled signaling cascades in mediating the glucagon increase induced by melatonin. In contrast, inhibition of phospholipase C in αTC1.9 cells prevented the melatonin-induced effect, indicating the physiological relevance of the Gαq-coupled pathway. Our data point to the involvement of the phosphatidylinositol 3-kinase signaling cascade in mediating melatonin effects in pancreatic α-cells. In conclusion, these findings provide evidence that the glucagon-stimulatory effect of melatonin in pancreatic α-cells is melatonin receptor mediated, thus supporting the concept of melatonin-modulated and diurnal glucagon release.

Phosphorylation of cyclic AMP-response element-binding protein (CREB) is influenced by melatonin treatment in pancreatic rat insulinoma ß-cells (INS-1).

The pineal hormone melatonin exerts its influence on the insulin secretion of pancreatic islets by a variety of signalling pathways. The purpose of the present study was to analyse the impact of melatonin on the phosphorylated transcription factor cAMP-response element-binding protein (pCREB). In pancreatic rat insulinoma ß-cells (INS-1), pCREB immunofluorescence intensities in cell nuclei using digitised confocal image analysis were measured to semi-quantify differences in the pCREB immunoreactivity (pCREB-ir) caused by different treatments. Increasing concentrations of forskolin or 3-isobutyl-1-methylxanthine (IBMX) resulted in a dose-dependent rise of the mean fluorescence intensity in pCREB-ir nuclear staining. Concomitant melatonin application significantly decreased pCREB-ir in INS-1 cells after 30-min, 1-hr and 3-hr treatment. The melatonin receptor antagonists luzindole and 4-phenyl-2-propionamidotetraline (4P-PDOT) completely abolished the pCREB phosphorylation-decreasing effect of melatonin, indicating that both melatonin receptor isoforms (MT(1) and MT(2)) are involved. In a transfected INS-1 cell line expressing the human MT(2) receptor, melatonin caused the greatest reduction in pCREB after IBMX treatment compared with nontransfected INS-1 cells, indicating a crucial influence of melatonin receptor density on pCREB regulation. Furthermore, the downregulation of pCREB by melatonin is concomitantly associated with a statistically significant downregulation of Camk2d transcript levels, as measured after 3 hr. In conclusion, the present study provides evidence that the phosphorylation level of CREB is modulated in pancreatic ß-cells by melatonin. Mediated via CREB, melatonin regulates the expression of genes that play an important functional role in the regulation of ß-cell signalling pathways.

The nervus intermedius as a variable landmark and critical structure in cerebellopontine angle surgery: an anatomical study and classification.

BACKGROUND: An understanding of the normal topography during cerebellopontine angle surgery is necessary to obviate the anatomical distortions caused by tumors. OBJECTIVE: The aim of this study was to analyze the morphological features of the nervus intermedius (NI) and its related structures in the cerebellopontine angle (CPA). METHODS: Forty-three isolated human brainstems were examined to collect comprehensive morphometric and topographical data of the NI in its course from the brainstem to the ganglion geniculi, and discover its anatomical relationship with the other neurovascular structures in the CPA as well as within the meatus acusticus internus. RESULTS: A total of 84 NI were analyzed. The number of bundles comprising the NI varied from one to five. The mean length of the cisternal segment of the NI was 11.47 mm. In most cases, a vein between the root entry/exit zones of the facial and the vestibulocochlear nerve (VN) was documented. In all cases the NI joined the facial nerve, typically (85 %) distally to the the porus within the meatus acusticus internus. The entry/exit zone of the NI can be categorized into four types: in type A, they arise directly from the brainstem; in type B, they arise solely from the facial nerve; in type C solely from the VN; and in type D, where the bundle or bundles arise from both the brainstem and the VN or the facial nerve. CONCLUSION: The anatomical features of the NI can provide an additional variable landmark and critical structure during cerebellopontine microsurgery. Our study of the nerve's anatomy and topographical relations may contribute to preventing intraoperative nerve injuries.

The central myelin-peripheral myelin transitional zone of the nervus intermedius and its implications for microsurgery in the cerebellopontine angle.

The central myelin-peripheral myelin transitional zone, also referred to as the "Obersteiner-Redlich zone (ORZ)" or "glial/Schwann junction" of the nervus intermedius, is thought to play a role in the pathophysiology of nervus intermedius neuralgia (NIN). To evaluate the location and histological features of the ORZ of the nervus intermedius (NI), 10 NI specimens from five fresh cadavers were microscopically analyzed for structural differences between their central and peripheral myelin segments. The ORZ was analyzed under a light microscope, and the exact location of the ORZ was confirmed by immunohistochemical staining using an oligodendroglial antibody. The total diameter of the NI showed a mean of 0.62 mm. The cisternal segment of the NI from the brain stem to the porus acusticus internus had a mean length of 13.97 mm. The mean extent of central myelin was 0.5 mm from the brain stem on the medial side and 0.33 mm on the lateral side. Moreover, the mean length of the ORZ was 0.279 mm on the medial side and 0.134 mm on the lateral side. The distance between the brain stem and the most distal point of central myelin that could be detected was 0.67 mm. Accordingly, the ORZ of the NI appears closer to the brain stem compared to the other cranial nerves. The exact location of the ORZ may play a role in diagnostic preoperative imaging, in the planning of surgical procedures for NIN, and may offer suitable landmarks for surgeons performing microvascular decompression in NIN treatment.

Human and Rodent Cell Lines as Models of Functional Melatonin-Responsive Pancreatic Islet Cells.

Cell culture of different pancreatic islet cell lines, like the murine α-cell line αTC1.9, the rat ß-cell lines INS-1 and INS-1 832/13, and the human δ-cell line QGP-1, can serve as valuable cell models for the analysis of melatonin-dependent modulation of hormone secretion. The paper summarizes in detail the requirements of culture for each cell line and includes batch protocols to stimulate hormone secretion and to treat cells with several melatonin concentrations as previously published. We here describe the processing of collected cell pellets or cell culture supernatants as well as different methods to analyze cell experiments after melatonin treatment on the basis of our own experience. Finally, we outlined for each cell line under which conditions the melatonin treatment should be performed to gain reproducible results.

Melatonin stimulates glucagon secretion in vitro and in vivo.

Recent investigations have demonstrated that melatonin influences carbohydrate metabolism mediated by insulin-inhibiting effects on pancreatic ß-cells. This study evaluated whether melatonin has also an effect on pancreatic α-cells and glucagon expression as well as the glucagon secretion in vitro and in vivo. Glucagon-producing pancreatic α-cell line αTC1 clone 9 (αTC1.9) was used, which was characterized as an appropriate model with glucose responsiveness and expression of the melatonin receptors MT1 and MT2. The results demonstrate that melatonin incubation significantly enhanced the expression as well as the secretion of glucagon. These effects appeared to be more pronounced under hyperglycemic conditions compared to basal glucose concentrations. Notably, in vivo studies demonstrated that long-term oral melatonin administration led to significantly elevated plasma glucagon concentrations in Wistar rats. In contrast, plasma glucagon levels were found to be slightly decreased in type 2 diabetic Goto-Kakizaki rats. Moreover, investigations measuring the relative glucagon receptor mRNA expression showed marked differences in the liver of melatonin-substituted rats as well as in melatonin receptor knockout mice. In conclusion, these findings revealed evidence that melatonin influences pancreatic glucagon expression and secretion as well as the peripheral glucagon action.

Melatonin inhibits insulin secretion in rat insulinoma ß-cells (INS-1) heterologously expressing the human melatonin receptor isoform MT2.

Melatonin exerts some of its effects via G-protein-coupled membrane receptors. Two membrane receptor isoforms, MT1 and MT2, have been described. The MT1 receptor is known to inhibit second messenger cyclic adenosine monophosphate (cAMP) signaling through receptor-coupling to inhibitory G-proteins (G(i) ). Much less is known about the MT2 receptor, but it has also been implicated in signaling via G(i) -proteins. In rat pancreatic ß-cells, it has recently been reported that the MT2 receptor plays an inhibitory role in the cyclic guanosine monophosphate (cGMP) pathway. This study addresses the signaling features of the constitutively expressed human recombinant MT2 receptor (hMT2) and its impact on insulin secretion, using a rat insulinoma ß-cell line (INS-1). On the basis of a specific radioimmunoassay, insulin secretion was found to be more strongly reduced in the clones expressing hMT2 than in INS-1 controls, when incubated with 1 or 100 nm melatonin. Similarly, cAMP and cGMP levels, measured by specific enzyme-linked immunosorbent assays (ELISAs), were reduced to a greater extent in hMT2 clones after melatonin treatment. In hMT2-expressing cells, the inhibitory effect of melatonin on insulin secretion was blocked by pretreatment with pertussis toxin, demonstrating the coupling of the hMT2 to G(i) -proteins. These results indicate that functional hMT2 expression leads to the inhibition of cyclic nucleotide signaling and a reduction in insulin release. Because genetic variants of the hMT2 receptor are considered to be risk factors in the development of type 2 diabetes, our results are potentially significant in explaining and preventing the pathogenesis of this disease.

Distribution patterns of calcium-binding proteins in pancreatic tissue of non-diabetic as well as type 2 diabetic rats and in rat insulinoma beta-cells (INS-1).

The present study dealt with the localization of different calcium-binding proteins (CaBPs) in the pancreatic tissue of non-diabetic and diabetic rats and in rat insulinoma beta-cells (INS-1). Transcripts of CaBPs displayed different expression levels in rat pancreatic tissue and INS-1 cells. Immunohistochemistry demonstrated that three of these proteins, calmodulin, calreticulin and calbindin-D28k, were located predominantly in the pancreatic islets (in both alpha- and beta-cells) of rats, showing weaker labeling of exocrine tissue. Secretagogin was exclusively found within islets. All CaBPs were also immunohistochemically detected in INS-1 cells. Immunohistochemical analysis demonstrates differences in CaBP distributions when comparing the pancreatic tissues of diabetic Goto-Kakizaki rats and non-diabetic Wistar rats. Pancreatic tissue in type 2 diabetic Goto-Kakizaki rats showed significantly higher transcript levels of all CaBPs compared to those in Wistar rats. These results indicate that alterations of CaBPs in pancreatic islets are associated with metabolic disturbances related to type 2 diabetes.

'TRPing' synaptic ribbon function in the rat pineal gland: neuroendocrine regulation involves the capsaicin receptor TRPV1.

Synaptic ribbons (SRs) are presynaptic structures thought to regulate and facilitate multivesicular release. In the pineal gland, they display a circadian rhythm with higher levels at night paralleling melatonin synthesis. To gain more insight into the processes involved and the possible functions of these structures, a series of experiments were conducted in rodents. We studied the regional distribution of a molecular marker of pineal SRs, the kinesin motor KIF3A in the gland. Respective immunoreactivity was abundant in central regions of the gland where sympathetic fibers were less dense, and vice versa, revealing that intercellular communication between adjacent pinealocytes is enhanced under low sympathetic influence. KIF3A was found to be colocalized to the transient receptor potential channel of the vanilloid receptor family, subtype 1 (TRPV1). The TRPV1 agonist capsaicin increased melatonin secretion from perifused pineals in a dose-dependent manner that was blocked by the competitive TRPV1 antagonist capsazepine. No change in free intracellular calcium was observed in response to TRPV1 ligands applied to pinealocytes responding to norepinephrine, bradykinin and/or depolarization. These data clearly indicate that TRPV1 actively regulates pineal gland function.

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.

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.

Modulation of the cGMP signaling pathway by melatonin in pancreatic beta-cells.

Melatonin influences the second messenger cyclic guanosine 3',5'-monophosphate (cGMP) signaling pathway in pancreatic beta-cells via a receptor-mediated mechanism. In the present study, it was determined how the regulation of cGMP concentrations by melatonin proceeds. The results provide evidence that melatonin acts via the soluble guanylate cyclase (sGC), as molecular investigations demonstrated that long-term incubation with melatonin significantly reduced the expression levels of the sGC mRNA in rat insulinoma beta-cells (INS1) cells, whereas mRNA expression of membrane guanylate cyclases was unaffected. Incubation with melatonin abolished the S-nitrosoacetyl penicillamine-induced increase of cGMP concentrations in INS1 cells. In addition, the cGMP-inhibitory effect of melatonin was reversed by preincubation with the sGC inhibitors 1H-(1,2,4)oxadiazolo(4,3-alpha)quinoxalin-1-one and 4H-8-bromo-1,2,4-oxadiazolo(3,4-d)benz(b)(1,4)oxazin-1-one. Nitric oxide (NO) production was not influenced after 1 hr of melatonin application, but was influenced after a 4 hr incubation period. Preincubation of INS1 cells with the NO synthase inhibitor N(G)-monomethyl-l-arginine did not abolish the cGMP-inhibitory effect of melatonin. Transcripts of cyclic nucleotide-gated (CNG) channels were significantly reduced after melatonin treatment in a dose-dependent manner, indicating the involvement of these channels in mediating the melatonin effect in INS1 cells. The results of this study demonstrate that melatonin mediates its inhibitory effect on cGMP concentrations in pancreatic beta-cells by inhibiting the sGC, but does not influence NO concentration or NO synthase activity in short-term incubation experiments. In addition, it was demonstrated that melatonin is involved in modulation of CNG channel mRNA.