Calorie restriction reduces pinealectomy-induced insulin resistance by improving GLUT4 gene expression and its translocation to the plasma membrane

The present study aimed to investigate insulin sensitivity and GLUT4 expression protein in pinealectomized rats, as well as to determining the effects of melatonin and calorie restriction on the changes induced by pinealectomy. Wistar rats were pinealectomized (Pinx) or sham operated (Sham), and studied 30 days later. Melatonin replacement treatment (50 g/100 g body weight) was continued for 30 days after pinealectomy. Calorie restriction was performed by offering 60% of the standard food intake. In vivo insulin sensitivity was evaluated using the glucose disappearance constant (kITT) during an insulin tolerance test, and GLUT4 mRNA and protein were assessed by Northern and Western blotting, respectively. The in vitro effect of melatonin on GLUT4 protein content in plasma membrane was investigated in adipocytes isolated from intact rats. Compared with Sham rats, Pinx rats showed decreased kITT (40%), GLUT4 expression in white adipose tissue (WAT, approximately 70%), and unchanged GLUT4 expression in skeletal muscle. Melatonin treatment in Pinx rats restored the kITT and GLUT4 protein to control values. No in vitro effects of melatonin (10-9 m) upon GLUT4 protein were observed. Calorie restriction of Pinx rats increased their kITT value ( approximately 40%), total GLUT4 protein content ( approximately 240%) and its translocation to the plasma membrane ( approximately 80%) in WAT. The results show that pinealectomy, for lack of melatonin, decreased insulin sensitivity as well as GLUT4 gene expression. Calorie restriction improved insulin sensitivity in Pinx rats, and this was related to increased GLUT4 gene expression and insulin-induced GLUT4 translocation to the plasma membrane in WAT.     

Melatonin effect on plasma adiponectin,leptin, insulin,glucose, triglycerides and cholesterol in normal and high fat–fed rats

Abstract: Melatonin effect on body weight progression, mean levels and 24‐hr pattern of circulating adiponectin, leptin, insulin, glucose, triglycerides and cholesterol were examined in rats fed a normal or a high‐fat diet. In experiment 1, rats fed a normal diet were divided into two groups: receiving melatonin (25 μg/mL drinking water) or vehicle for 9 wk. In experiment 2, animals were divided into three groups: two fed with a high‐fat diet (35% fat) and melatonin (25 μg/mL) or vehicle in drinking water for 11 wk, while a third group was given a normal diet (4% fat). At the end of experiments, groups of eight rats were killed at six different time intervals throughout a 24‐ hr period. Melatonin administration for 9 wk decreased body weight gain from the 3rd wk on without affecting food intake. A significant reduction in circulating insulin, glucose and triglyceride mean levels and disrupted daily patterns of plasma adiponectin, leptin and insulin were observed after melatonin. In high fat–fed rats, melatonin attenuated body weight increase, hyperglycemia and hyperinsulinemia, as well as the increase in mean plasma adiponectin, leptin, triglycerides and cholesterol levels. The high‐fat diet disrupted normal 24‐ hr patterns of circulating adiponectin, insulin and cholesterol, the effects on insulin and cholesterol being counteracted by melatonin. Nocturnal plasma melatonin concentration in control and obese rats receiving melatonin for 11 wk attained values 21–24‐fold greater than controls. The results indicate that melatonin counteracts some of the disrupting effects of diet‐induced obesity in rats.     

Melatonin improves insulin sensitivity independently of weight loss in old obese rats

In aged rats, insulin signaling pathway (ISP) is impaired in tissues that play a pivotal role in glucose homeostasis, such as liver, skeletal muscle, and adipose tissue. Moreover, the aging process is also associated with obesity and reduction in melatonin synthesis from the pineal gland and other organs. The aim of the present work was to evaluate, in male old obese Wistar rats, the effect of melatonin supplementation in the ISP, analyzing the total protein amount and the phosphorylated status (immunoprecipitation and immunoblotting) of the insulin cascade components in the rat hypothalamus, liver, skeletal muscle, and periepididymal adipose tissue. Melatonin was administered in the drinking water for 8‐ and 12 wk during the night period. Food and water intake and fasting blood glucose remained unchanged. The insulin sensitivity presented a 2.1‐fold increase both after 8‐ and 12 wk of melatonin supplementation. Animals supplemented with melatonin for 12 wk also presented a reduction in body mass. The acute insulin‐induced phosphorylation of the analyzed ISP proteins increased 1.3‐ and 2.3‐fold after 8‐ and 12 wk of melatonin supplementation. The total protein content of the insulin receptor (IR) and the IR substrates (IRS‐1, 2) remained unchanged in all investigated tissues, except for the 2‐fold increase in the total amount of IRS‐1 in the periepididymal adipose tissue. Therefore, the known age‐related melatonin synthesis reduction may also be involved in the development of insulin resistance and the adequate supplementation could be an important alternative for the prevention of insulin signaling impairment in aged organisms.     

Chronic melatonin consumption prevents obesity-related metabolic abnormalities and protects the heart against myocardial ischemia and reperfusion injury in a prediabetic model of diet-induced obesity

Abstract: Obesity, a major risk factor for ischemic heart disease, is associated with increased oxidative stress and reduced antioxidant status. Melatonin, a potent free radical scavenger and antioxidant, has powerful cardioprotective effects in lean animals but its efficacy in obesity is unknown. We investigated the effects of chronic melatonin administration on the development of the metabolic syndrome as well as ischemia–reperfusion injury in a rat model of diet‐induced obesity (DIO). Male Wistar rats received a control diet, a control diet with melatonin, a high‐calorie diet, or a high‐calorie diet with melatonin (DM). Melatonin (4 mg/kg/day) was administered in the drinking water. After 16 wk, biometric and blood metabolic parameters were measured. Hearts were perfused ex vivo for the evaluation of myocardial function, infarct size (IFS) and biochemical changes [activation of PKB/Akt, ERK, p38 MAPK, AMPK, and glucose transporter (GLUT)‐4 expression). The high‐calorie diet caused increases in body weight (BW), visceral adiposity, serum insulin and triglycerides (TRIG), with no change in glucose levels. Melatonin treatment reduced the BW gain, visceral adiposity, blood TRIG, serum insulin, homeostatic model assessment index and thiobarbituric acid reactive substances in the DIO group. Melatonin reduced IFS in DIO and control groups and increased percentage recovery of functional performance of DIO hearts. During reperfusion, hearts from melatonin‐treated rats had increased activation of PKB/Akt, ERK42/44 and reduced p38 MAPK activation. Chronic melatonin treatment prevented the metabolic abnormalities induced by DIO and protected the heart against ischemia–reperfusion injury. These beneficial effects were associated with activation of the reperfusion injury salvage kinases pathway.     

Short‐term melatonin consumption protects the heart of obese rats independent of body weight change and visceral adiposity

Chronic melatonin treatment has been shown to prevent the harmful effects of diet‐induced obesity and reduce myocardial susceptibility to ischaemia‐reperfusion injury (IRI). However, the exact mechanism whereby it exerts its beneficial actions on the heart in obesity/insulin resistance remains unknown. Herein, we investigated the effects of relatively short‐term melatonin treatment on the heart in a rat model of diet‐induced obesity. Control and diet‐induced obese Wistar rats (fed a high calorie diet for 20 wk) were each subdivided into three groups receiving drinking water with or without melatonin (4 mg/kg/day) for the last 6 or 3 wk of experimentation. A number of isolated hearts were perfused in the working mode, subjected to regional or global ischaemia‐reperfusion; others were nonperfused. Metabolic parameters, myocardial infarct sizes (IFS), baseline and postischaemic activation of PKB/Akt, ERK42/44, GSK‐3β and STAT‐3 were determined. Diet‐induced obesity caused increases in body weight gain, visceral adiposity, fasting blood glucose, serum insulin and triglyceride (TG) levels with a concomitant cardiac hypertrophy, large postischaemic myocardial IFSs and a reduced cardiac output. Melatonin treatment (3 and 6 wk) decreased serum insulin levels and the HOMA index (P < 0.05) with no effect on weight gain (after 3 wk), visceral adiposity, serum TG and glucose levels. It increased serum adiponectin levels, reduced myocardial IFSs in both groups and activated baseline myocardial STAT‐3 and PKB/Akt, ERK42/44 and GSK‐3β during reperfusion. Overall, short‐term melatonin administration to obese/insulin resistant rats reduced insulin resistance and protected the heart against ex vivo myocardial IRI independently of body weight change and visceral adiposity.     

Melatonin, endocrine pancreas and diabetes

Melatonin influences insulin secretion both in vivo and in vitro. (i) The effects are MT(1)-and MT(2)-receptor-mediated. (ii) They are specific, high-affinity, pertussis-toxin-sensitive, G(i)-protein-coupled, leading to inhibition of the cAMP-pathway and decrease of insulin release. [Correction added after online publication 4 December 2007: in the preceding sentence, 'increase of insulin release' was changed to 'decrease of insulin release'.] Furthermore, melatonin inhibits the cGMP-pathway, possibly mediated by MT(2) receptors. In this way, melatonin likely inhibits insulin release. A third system, the IP(3)-pathway, is mediated by G(q)-proteins, phospholipase C and IP(3), which mobilize Ca(2+) from intracellular stores, with a resultant increase in insulin. (iii) Insulin secretion in vivo, as well as from isolated islets, exhibits a circadian rhythm. This rhythm, which is apparently generated within the islets, is influenced by melatonin, which induces a phase shift in insulin secretion. (iv) Observation of the circadian expression of clock genes in the pancreas could possibly be an indication of the generation of circadian rhythms in the pancreatic islets themselves. (v) Melatonin influences diabetes and associated metabolic disturbances. The diabetogens, alloxan and streptozotocin, lead to selective destruction of beta-cells through their accumulation in these cells, where they induce the generation of ROS. Beta-cells are very susceptible to oxidative stress because they possess only low-antioxidative capacity. Results suggest that melatonin in pharmacological doses provides protection against ROS. (vi) Finally, melatonin levels in plasma, as well as the arylalkylamine-N-acetyltransferase (AANAT) activity, are lower in diabetic than in nondiabetic rats and humans. In contrast, in the pineal gland, the AANAT mRNA is increased and the insulin receptor mRNA is decreased, which indicates a close interrelationship between insulin and melatonin.     

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.     

Aging-dependent changes in the effect of daily melatonin supplementation on rat metabolic and behavioral responses

Pineal melatonin secretion has been reported to commonly decrease with aging, whereas intra-abdominal adiposity, plasma insulin and plasma leptin levels tend to increase. We recently demonstrated that daily melatonin administration starting at middle age suppressed male rat intra-abdominal fat, plasma leptin and plasma insulin to youthful levels, suggesting that aging-related changes in pineal melatonin secretion and in energy regulation may be functionally related. Accordingly, we have now investigated the effects of daily melatonin treatment on energy regulation in young versus middle-aged male Sprague Dawley rats. Addition of melatonin to the drinking water (0.2 microg/mL) produced nocturnal and diurnal plasma melatonin concentrations in middle-aged rats (12 months) equivalent to those of young adult (5 months) rats. Administration of this melatonin dosage every day for 10 wk starting at 10 months of age suppressed (P < 0.01) relative intra-abdominal fat, non-fasted plasma insulin and plasma leptin by 27, 39, and 51%, respectively (vs. vehicle-treated controls). In contrast, administration of melatonin for 10 wk starting at 3 months of age did not significantly alter (P> 0.10) any of these parameters. The melatonin administration stimulated (102%, P < 0.001) behavioral responsiveness of the middle-aged rats in a test of response to novelty, restoring youthful levels, but did not significantly alter behavioral responsiveness of the young rats. These results suggest that suppression of intra-abdominal adiposity and plasma leptin and insulin levels and stimulation of behavioral responsiveness in response to daily exogenous melatonin begins at middle age, coincident with and likely dependent upon the aging-associated decline in endogenous pineal melatonin secretion. These results further suggest that appropriate melatonin supplementation may potentially provide therapy or prophylaxis not only for the insulin resistance, increased intra-abdominal fat and resulting pathologies that occur with aging, but also for some aging-associated behavioral changes.     

Melatonin improves insulin resistance and hepatic steatosis through attenuation of alpha‐2‐HS‐glycoprotein

Melatonin plays an important role in regulating circadian rhythms. It also acts as a potent antioxidant and regulates glucose and lipid metabolism, although the exact action mechanism is not clear. The α2‐HS‐glycoprotein gene (AHSG) and its protein, fetuin‐A (FETUA), are one of the hepatokines and are known to be associated with insulin resistance and type 2 diabetes. The aim of this study was to determine whether melatonin improves hepatic insulin resistance and hepatic steatosis in a FETUA‐dependent manner. In HepG2 cells treated with 300 μmol/L of palmitic acid, phosphorylated AKT expression decreased, and FETUA expression increased, but this effect was inhibited by treatment with 10 μmol/L of melatonin. However, melatonin did not improve insulin resistance in FETUA‐overexpressing cells, indicating that improvement in insulin resistance by melatonin was dependent on downregulation of FETUA. Moreover, melatonin decreased palmitic acid‐induced ER stress markers, CHOP, Bip, ATF‐6, XBP‐1, ATF‐4, and PERK. In addition, in the high‐fat diet (HFD) mice, oral treatment with 100 mg/kg/day melatonin for 10 weeks reduced body weight gain to one‐third of that of the HFD group and hepatic steatosis. Insulin sensitivity and glucose intolerance improved with the upregulation of muscle p‐AKT protein expression. FETUA expression and ER stress markers in the liver and serum of HFD mice were decreased by melatonin treatment. In conclusion, melatonin can improve hepatic insulin resistance and hepatic steatosis through reduction in ER stress and the resultant AHSG expression.     

Adaptations of the aging animal to exercise: role of daily supplementation with melatonin

The pineal gland, through melatonin, seems to be of fundamental importance in determining the metabolic adaptations of adipose and muscle tissues to physical training. Evidence shows that pinealectomized animals fail to develop adaptive metabolic changes in response to aerobic exercise and therefore do not exhibit the same performance as control‐trained animals. The known prominent reduction in melatonin synthesis in aging animals led us to investigate the metabolic adaptations to physical training in aged animals with and without daily melatonin replacement. Male Wistar rats were assigned to four groups: sedentary control (SC), trained control (TC), sedentary treated with melatonin (SM), and trained treated with melatonin (TM). Melatonin supplementation lasted 16 wk, and the animals were subjected to exercise during the last 8 wk of the experiment. After euthanasia, samples of liver, muscle, and adipose tissues were collected for analysis. Trained animals treated with melatonin presented better results in the following parameters: glucose tolerance, physical capacity, citrate synthase activity, hepatic and muscular glycogen content, body weight, protein expression of phosphatidylinositol 3‐kinase (PI3K), mitogen‐activated protein kinase (MAPK), and protein kinase activated by adenosine monophosphate (AMPK) in the liver, as well as the protein expression of the glucose transporter type 4 (GLUT4) and AMPK in the muscle. In conclusion, these results demonstrate that melatonin supplementation in aging animals is of great importance for the required metabolic adaptations induced by aerobic exercise. Adequate levels of circulating melatonin are, therefore, necessary to improve energetic metabolism efficiency, reducing body weight and increasing insulin sensitivity.     

Melatonin,energy metabolism,and obesity: a review

Melatonin is an old and ubiquitous molecule in nature showing multiple mechanisms of action and functions in practically every living organism. In mammals, pineal melatonin functions as a hormone and a chronobiotic, playing a major role in the regulation of the circadian temporal internal order. The anti‐obesogen and the weight‐reducing effects of melatonin depend on several mechanisms and actions. Experimental evidence demonstrates that melatonin is necessary for the proper synthesis, secretion, and action of insulin. Melatonin acts by regulating GLUT4 expression and/or triggering, via its G‐protein‐coupled membrane receptors, the phosphorylation of the insulin receptor and its intracellular substrates mobilizing the insulin‐signaling pathway. Melatonin is a powerful chronobiotic being responsible, in part, by the daily distribution of metabolic processes so that the activity/feeding phase of the day is associated with high insulin sensitivity, and the rest/fasting is synchronized to the insulin‐resistant metabolic phase of the day. Furthermore, melatonin is responsible for the establishment of an adequate energy balance mainly by regulating energy flow to and from the stores and directly regulating the energy expenditure through the activation of brown adipose tissue and participating in the browning process of white adipose tissue. The reduction in melatonin production, as during aging, shift‐work or illuminated environments during the night, induces insulin resistance, glucose intolerance, sleep disturbance, and metabolic circadian disorganization characterizing a state of chronodisruption leading to obesity. The available evidence supports the suggestion that melatonin replacement therapy might contribute to restore a more healthy state of the organism.     

Melatonin can improve insulin resistance and aging-induced pancreas alterations in senescence-accelerated prone male mice (SAMP8)

The aim of the present study was to investigate the effect of aging on several parameters related to glucose homeostasis and insulin resistance in pancreas and how melatonin administration could affect these parameters. Pancreas samples were obtained from two types of male mice models: senescence-accelerated prone (SAMP8) and senescence-accelerated-resistant mice (SAMR1). Insulin levels in plasma were increased with aging in both SAMP8 and SAMR1 mice, whereas insulin content in pancreas was decreased with aging in SAMP8 and increased in SAMR1 mice. Expressions of glucagon and GLUT2 messenger RNAs (mRNAs) were increased with aging in SAMP8 mice, and no differences were observed in somatostatin and insulin mRNA expressions. Furthermore, aging decreased also the expressions of Pdx-1, FoxO 1, FoxO 3A and Sirt1 in pancreatic SAMP8 samples. Pdx-1 was decreased in SAMR1 mice, but no differences were observed in the rest of parameters on these mice strains. Treatment with melatonin was able to decrease plasma insulin levels and to increase its pancreatic content in SAMP8 mice. In SAMR1, insulin pancreatic content and plasma levels were decreased. HOMA-IR was decreased with melatonin treatment in both strains of animals. On the other hand, in SAMP8 mice, treatment decreased the expression of glucagon, GLUT2, somatostatin and insulin mRNA. Furthermore, it was also able to increase the expression of Sirt1, Pdx-1 and FoxO 3A. According to these results, aging is associated with significant alterations in the relative expression of pancreatic genes associated to glucose metabolism. This has been especially observed in SAMP8 mice. Melatonin administration was able to improve pancreatic function in old SAMP8 mice and to reduce HOMA-IR improving their insulin physiology and glucose metabolism.     

Immunohistochemical Assessment of Melatonin Binding in the Pineal Gland

Melatonin binding in the pineal gland of albino rats is estimated using an immunohistochemical procedure. Binding is saturable, has relatively high affinity (Apparent KD = 2.7 nM), and competition studies indicate binding of indoleamines possessing an N-acetyl group on the terminus of the side chain (N-acetylserotonin and melatonin). These data are consistent with the interpretation that immunohistochemically determined melatonin in unfixed pineal tissue is assessing binding of N-acetylated indolealkylamines to pineal cell components. In albino rats maintained on 12-hour light: 12-hour dark cycles, melatonin binding exhibits a diurnal rhythm with low levels of saturation (30%) early in the light and saturation by endogenous melatonin near the onset of darkness. An annual rhythm of melatonin binding was observed in albino rats with low levels during the summer and high levels during the winter. Other rats were maintained on 12-hour light:dark cycles and fed for 2 hours either early in the light period or early in the dark period. For both morning- and evening-fed animals, melatonin binding was high prior to feeding and dropped immediately after feeding. Changes in melatonin binding that occur in response to alterations of feeding and time of year suggest the possibility that this binding reflects a functional site for melatonin.     

Melatonin prevents mitochondrial dysfunction and insulin resistance in rat skeletal muscle

Melatonin has a number of beneficial metabolic actions and reduced levels of melatonin may contribute to type 2 diabetes. The present study investigated the metabolic pathways involved in the effects of melatonin on mitochondrial function and insulin resistance in rat skeletal muscle. The effect of melatonin was tested both in vitro in isolated rats skeletal muscle cells and in vivo using pinealectomized rats (PNX). Insulin resistance was induced in vitro by treating primary rat skeletal muscle cells with palmitic acid for 24 hr. Insulin‐stimulated glucose uptake was reduced by palmitic acid followed by decreased phosphorylation of AKT which was prevented my melatonin. Palmitic acid reduced mitochondrial respiration, genes involved in mitochondrial biogenesis and the levels of tricarboxylic acid cycle intermediates whereas melatonin counteracted all these parameters in insulin‐resistant cells. Melatonin treatment increases CAMKII and p‐CREB but had no effect on p‐AMPK. Silencing of CREB protein by siRNA reduced mitochondrial respiration mimicking the effect of palmitic acid and prevented melatonin‐induced increase in p‐AKT in palmitic acid‐treated cells. PNX rats exhibited mild glucose intolerance, decreased energy expenditure and decreased p‐AKT, mitochondrial respiration, and p‐CREB and PGC‐1 alpha levels in skeletal muscle which were restored by melatonin treatment in PNX rats. In summary, we showed that melatonin could prevent mitochondrial dysfunction and insulin resistance via activation of CREB‐PGC‐1 alpha pathway. Thus, the present work shows that melatonin play an important role in skeletal muscle mitochondrial function which could explain some of the beneficial effects of melatonin in insulin resistance states.     

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.     

Evidence of the receptor‐mediated influence of melatonin on pancreatic glucagon secretion via the Gαq protein‐coupled and PI3K signaling pathways

Abstract: 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.