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Melatonin increases intracellular calcium in the liver, muscle, white adipose tissues and pancreas of diabetic obese rats A. Agil,*a E. K. Elmahallawy,a J. M. Rodríguez-Ferrer,b A. Adem,c S. M. Bastaki,c I. Al-abbadi,d Y. A. Fino Solanoa,e and M. Navarro-Alarcón*e Melatonin, a widespread substance with antioxidant and anti-inflammatory properties, has been found to act as an antidiabetic agent in animal models, regulating the release and action of insulin. However, the molecular bases of this antidiabetic action are unknown, limiting its application in humans. Several studies have recently shown that melatonin can modify calcium (Ca2+) in diabetic animals, and Ca2+ has been reported to be involved in glucose homeostasis. The objective of the present study was to assess whether the antidiabetic effect of chronic melatonin at pharmacological doses is established via Ca2+ regulation in different tissues in an animal model of obesity-related type 2 diabetes, using Zücker diabetic fatty (ZDF) rats and their lean littermates, Zücker lean (ZL) rats. After the treatments, flame atomic absorption spectrometry was used to determine Ca2+ levels in the liver, muscle, main types of internal white adipose tissue, subcutaneous lumbar fat, pancreas, brain, and plasma. This study reports for the first time that chronic melatonin administration (10 mg per kg body weight per day for 6 weeks) increases Ca2+ levels in muscle, liver, different adipose tissues, and pancreas in ZDF rats, although there were no significant
Received 22nd May 2015, Accepted 23rd June 2015
changes in their brain or plasma Ca2+ levels. We propose that this additional peripheral dual action
DOI: 10.1039/c5fo00590f
mechanism underlies the improvement in insulin sensitivity and secretion previously documented in samples from the same animals. According to these results, indoleamine may be a potential candidate for
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the treatment of type 2 diabetes mellitus associated with obesity.
Introduction Melatonin, N-acetyl-5-methoxytryptamine, is an indoleamine mainly produced in peripheral organs and tissues in larger amounts than in the pineal gland, although pineal, but not extrapineal melatonin, retains chronobiotic properties.1–5 The widespread presence of melatonin and its variety of biological properties suggest that it is a very ancient molecule with important roles in phylogenetic distant organisms.6 Various researchers have described this indolamine as a highly interesting option for maintaining optimal health conditions.2,7–9 Melatonin has been found to exert an antioxidant effect via
a Department of Pharmacology and Neurosciences Institute (CIBM), School of Medicine, University of Granada, Spain. E-mail: [email protected]; Fax: +34958243537 b Department of Physiology and Neurosciences Institute (CIBM), School of Medicine, University of Granada, Spain c Department of Pharmacology, School of Medicine and Health Sciences, University of United Arab Emirates, Al Ain, United Arab Emirates d 1678 School of Pharmacy, University of Jordan, Amman, Jordan e Department of Nutrition and Food Science, School of Pharmacy, University of Granada, Spain
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different mechanisms,7,8,10–12 confirming its role in the control of inflammation and oxidative stress, which underlie various chronic diseases.13–18 Some authors have proposed that melatonin may be useful against the increasing pandemic of overweight, obesity and obesity-related diabetes worldwide.19 These reports are in the same line as previous observations by our group that chronic melatonin administration improves diabetes by ameliorating the homeostatic model assessment insulin resistance (HOMA-IR) and homeostasis model assessment β-cell functionality (HOMA-IH),16 and counteracts oxidative stress and low-grade inflammation in the ZDF diabetes/obesity model.16 Furthermore, several studies have demonstrated that age-related insulin secretion dysfunction plays an important role in glucose metabolism disorders at older ages, contributing to the high indexes of glucose intolerance in elderly populations.17,18 Calcium (Ca2+) is a controlling messenger in eukaryotic cell signaling and is involved in a plethora of events in different biological models.20 Ca2+ was found to play a key role in β-cell functionality,21 and a lower intracellular level of Ca2+ was associated with the presence of hyperglycemia and a depolarizing K+ stimulus.22 These effects may also be related to a poss-
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ible alteration of the adenosine triphosphate (ATP)-sensitive K+ channel (KATP channel). Hence, a decrease in Ca2+ regulation and glucose signaling appears to contribute to the reduction in β-cell stimulus-insulin secretion coupling in rats of an advanced age.23 Additionally, HOMA-IR studies in key tissues revealed a relationship between insulin tolerance and adequate Ca2+ levels in muscle cells.22 Glucose transporter type 4 (GLUT4) is known to be highly expressed in adipose tissue, skeletal, muscle and liver.24,25 Two cellular events that contribute to the improvement of GLUT4 translocation to the cell surface have been observed during muscle contraction: a transient increase in intracellular Ca2+ concentration ([Ca2+]i) and elevation of the [ATP]/[AMP] ratio.25–27 This process is mediated by the activation of the Ca2+/calmodulin-dependent protein kinase (CaMKII), which is also required for Ca2+ homeostasis and its reuptake.28,29 It has also been reported that a calcium ionophore-induced rise in cytosolic calcium produces an increase in glucose transporter type 1 (GLUT1) protein and mRNA, which enhances the entry of glucose.6,30 We have found no published study on the effect of melatonin supplementation on the regulation of Ca2+ levels in tissues. Our group previously reported that melatonin administrations improve various biomarkers associated with type 2 diabetes mellitus (T2DM) through its effect on chromium (Cr) and vanadium (V).16,17,31,32 Importantly, melatonin also affects CaMKII protein levels, probably acting indirectly via the Ca2+calmodulin signaling pathway for regulating insulin secretion.33 Hence, melatonin can enhance the capacity of β cells to respond to metabolic signals by impacting on this kinase group.34 With this background, the objective of the present study was to investigate the effects of chronic melatonin administration on Ca2+ homeostasis in liver, muscle, the main types of internal white adipose tissues (visceral omentum, gonadal and renal), subcutaneous lumbar fat, pancreas, brain, pancreas and plasma in young male ZDF rats and their lean littermates (ZL rats).
Results and discussion As shown in Fig. 1–3, the four subgroups significantly differed (P < 0.05) in Ca2+ levels measured in the liver, muscle, pancreas, WAT [visceral omental, gonadal and renal] and lumbar subcutaneous fat samples but not in those measured in brain or plasma samples (P > 0.05). The ZDF-M group showed a significantly higher mean Ca2+ level in: liver versus ZDF-C, ZL-C and ZL-M groups (0.185 ± 0.031 vs. 0.073 ± 0.023, 0.115 ± 0.024 and 0.070 ± 0.040 mg g−1, respectively; P < 0.05; Fig. 1A); muscle versus ZDF-C and ZL-C groups (0.248 ± 0.033 vs. 0.095 ± 0.033 and 0.131 ± 0.022 mg g−1, respectively; P < 0.05; Fig. 1B); and gonadal fat versus the ZDF-C, ZL-C and ZL-M groups (0.876 ± 0.217 vs. 0.239 ± 0.123, 0.042 ± 0.015 and 0.052 ± 0.019 mg g−1, respectively, P < 0.01). Higher mean Ca2+ levels were found in the omental white fat of the ZDF-M and ZL-M
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groups versus the ZDF-C group (0.201 ± 0.020 and 0.160 ± 0.015 vs. 0.081 ± 0.015 mg g−1, respectively, P < 0.05 Fig. 2A and B), and the ZDF-M group showed a significantly higher mean Ca2+ level versus the ZDF-C group in renal visceral WAT (from 0.064 ± 0.040 to 0.201 ± 0.026 mg g−1, P < 0.05; Fig. 2C), lumbar subcutaneous fat (from 0.093 ± 0.068 to 0.223 ± 0.055 mg g−1, P < 0.01 Fig. 2D) and pancreas (from 0.194 ± 0.091 to 0.336 ± 0.030 mg g−1, P < 0.05; Fig. 1C). No significant differences in brain (Fig. 1D) or plasma (Fig. 3) Ca2+ levels were found among the four study subgroups (P > 0.05). No significant differences were found in the mean Ca2+ levels of any tissue (including plasma) between the ZDF-C and ZL-C groups, as exhibited in the figures, or between the control and vehicle-treated animals (data not shown). In this study, the chronic administration of melatonin during 6 weeks in diabetic obese ZDF rats at a dose within the pharmacological range (10 mg per kg body weight per day) increased their intracellular Ca2+ levels in the liver, muscle and pancreas but had no effect on their brain tissue or plasma Ca2+ concentrations in ZDF rats. These findings add to evidence previously obtained in samples from the same animal on the positive effects of melatonin on fasting glucose and insulin blood levels (β-cell function, as estimated by HOMA1%B index), insulin resistance (HOMA-IR index), and the leptin/ adiponectin ratio.16,31 These findings together with the great role played by melatonin in the reduction of free fatty acid and oxidative or nitrosative stress emphasize the beneficial effect of melatonin treatment on age related insulin resistance and on the development of T2DM.18 As noted above, melatonin administration can increase plasma levels of V and Cr, which are both related to glucose tolerance,32 and it can reduce the body weight of ZDF rats for the same food intake, related to an improved lipid profile.31 The relationship between melatonin and Ca2+ flux regulation has been studied in numerous cells.35–37 In pancreatic β cells, hyperglycemia and hyperlipidemia were reported to reduce insulin sensitivity by hindering Ca2+ entry into the cell due to the high [AMP]/[ATP] ratio.22 Other researchers found that melatonin increased intracellular Ca2+ in macrophages of streptozotocin-induced diabetic rats.38 In addition, the hyperglycemic and hyperlipidemic characteristics of T2DM in proopiomelanocortin (POMC) system cells and pancreatic β cells have been associated with an increase in superoxide radicals and therefore in the expression of protein uncoupling protein2 (UCP2). This would reduce ATP production and consequently the ATP/ADP ratio.22 Hence, there is a reduced expression in fatty rats of the ATP-dependent K+ channel (KATP channel) component, designated Kir6.2 protein (Potassium Channel, Inwardly Rectifying, Kir6.2), which hinders K+ channel closure.33 As a result, the voltage-sensitive calcium channel (VSCC) system is not blocked, ultimately impeding Ca2+ entry into the cell, which facilitates insulin sensitivity loss and exacerbates T2DM. It should be remembered that the ADP/ATP ratio can be reduced by increased ATP synthesis, favoring the closure of the KATP kir6.2 channel, opening the cell membrane Ca2+ channel, increasing intracellular Ca2+, improving
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Fig. 1 (A) Effect of melatonin treatment on calcium levels (mg g−1) in the liver. Values are means ± SEM ZDF, Zücker Diabetic Fatty rats; ZL, Zücker Lean rats. *P < 0.05, **P < 0.01. (B) Effect of melatonin treatment on calcium levels (mg g−1) in skeletal muscle. Values are means ± SEM ZDF, Zücker Diabetic Fatty rats; ZL, Zücker Lean rats. ***P < 0.001. (C) Effect of melatonin treatment on calcium levels (mg g−1) in the pancreas. Values are means ± SEM ZDF, Zücker Diabetic Fatty rats; ZL, Zücker Lean rats. **P < 0.01. (D) Effect of melatonin treatment on calcium levels (mg g−1) in the brain. Values are means ± SEM ZDF, Zücker Diabetic Fatty rats; ZL, Zücker Lean rats.
glucose signaling and reducing insulin resistance, while calcium signaling in mitochondria stimulates ATP production.22,39 The major role played by melatonin at the subcellular level can be largely attributed to its dual effect on mitochondrial permeability transition pore (mPTP) opening, the gatekeeper of apoptotic and necrotic cell death, which consequently controls Ca2+ homeostasis.37,40 In this line, it was recently observed that chronic melatonin administrations markedly inhibit Ca2+-induced mPTP opening in mitochondria isolated from white and beige fat of both diabetic fatty and lean rats.41 Melatonin may contribute via these mechanisms to decrease oxidative stress and therefore reduce the induction of
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mPTP opening, maintaining intramitochondrial Ca2+ levels. However, the increased Ca2+ in the studied tissues, especially in WAT [visceral omental, gonadal and renal] and lumbar subcutaneous fat samples, does not imply an increase in mitochondrial levels, because melatonin was found to inhibit Ca2+induced mPTP opening in isolated mitochondria,41 but it is associated with the decreased oxidative stress described in previous studies using the same animal model.17,41 It should be taken into account that the calcium-activation of mPTP is inhibited by the overexpression of antiapoptotic B-cell lymphoma 2 (bcl-2),40 which is induced by melatonin treatment in liver,42 heart43 and brain,44 among other tissues.
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Fig. 2 (A) Effect of melatonin treatment on calcium levels (mg g−1) in omental white adipose tissue. Values are means ± SEM ZDF, Zücker Diabetic Fatty rats; ZL, Zücker Lean rats. *P < 0.05, **P < 0.01. (B) Effect of melatonin treatment on calcium levels (mg g−1) in gonadal white adipose tissue. Values are means ± SEM ZDF, Zücker Diabetic Fatty rats; ZL, Zücker Lean rats. *P < 0.05, ***P < 0.001. (C) Effect of melatonin treatment on calcium levels (mg g−1) in renal white adipose tissue. Values are means ± SEM ZDF, Zücker Diabetic Fatty rats; ZL, Zücker Lean rats. *P < 0.05. (D) Effect of melatonin treatment on calcium levels (mg g−1) in subcutaneous fat. Values are means ± SEM ZDF, Zücker Diabetic Fatty rats; ZL, Zücker Lean rats. **P < 0.01.
The liver is considered the main organ involved in maintaining normal glucose homeostasis through its role in regulating hepatic gluconeogenesis and glycolysis.45,46 As depicted in Fig. (1A), liver Ca2+ levels were higher in melatonin-treated ZDF rats than in the other groups (ZDF-C, ZL-M and ZL-C), which may also be related to an improved glucose tolerance, in common with previous studies which showed that melatonin is very useful in the protection of steatotic livers in ZDF rats which is not found in ZL rats.46,47 The higher levels of Ca2+ in melatonin-treated ZDF rats are consistent with various reports of its association with a decrease in insulin resistance.22,31,32 In humans, GLUT4
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protein is encoded by the GLUT4 gene and is a well-known insulin-regulated glucose transporter.24,25 It is highly expressed in adipose tissues and striated muscle and is closely related to glucose levels.48–50 Muscle contraction transiently increases the intracellular cytoplasmic concentration of Ca2+ with a concomitant elevation of the AMP/ATP ratio,51,52 which improves the glucose uptake of GLUT4 and glucose tolerance observed in this animal model of obesity and diabetes.31 Therefore, melatonin might indirectly enhance the externalization of the GLUT-4 receptor, improving glucose homeostasis in the tissues in which Ca2+ levels were increased. Contrarily, it has been lately reported that melatonin reduces the uptake of
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Fig. 3 Effect of melatonin treatment on calcium levels (mg ml−1) in plasma. Values are means ± SEM ZDF, Zücker Diabetic Fatty rats; ZL, Zücker Lean rats.
glucose and modifies the expression of GLUT1 transported in prostate cancer cells.6 Melatonin-treated ZDF rats also showed significantly higher Ca2+ levels in comparison to untreated ZDF-C rats in all adipose tissue samples (WAT [visceral omental, gonadal and renal] and lumbar subcutaneous fat). In obesity-related T2DM, adipose tissue was reported to actively participate in metabolic homeostasis through its capacity to produce adipocytokines, including leptin and adiponectin.31,53 Leptin is produced by mature adipocytes that have been hypertrophied due to fat accumulation. Most studies have observed elevated blood leptin levels (hyperleptinemia) in ZDF rats, attributable to their strong genetic resistance to this adipocytokine through the homozygous mutation of its receptor, which therefore has virtually no signaling capacity. This situation produces hyperphagia and reduced energy expenditure, resulting in severe obesity.54 Various authors have observed a reduction in leptin levels after melatonin administration.55–58 It has also been reported that melatonin-induced weight loss may improve the adipocyte metabolism and thereby increase the secretion of adiponectin, whose levels are significantly reduced in fatty animals.31 This finding reported by our research group was in samples from the same ZDF rats where melatonin raised hypoadiponectinemia by 40% and concomitantly decreased hyperleptinemia by 34%.16 Circulating adiponectin levels are generally inversely correlated with fatty mass and insulin resistance, being lower in obesity and T2DM in both animals and humans.59 This is of special relevance, given that adiponectin is an insulin sensitizer60 in the liver, muscle and peripheral tissues,61 and low levels increase the risk of T2DM.60 Adiponectin modulation may be a potential therapeutic strategy by using certain Ca2+ channel blockers to enhance its levels, which may help to suppress or prevent the onset of diseases related to inflammatory processes, such as atherogenesis.60 In the present study, melatonin administration increased the levels of Ca2+ in all studied adipose tissues (white omental, gonadal, renal and lumbar subcutaneous fat),
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and its metabolism is considerably altered in obesity and diabetes. The Ca2+ increase observed in the present adipose tissue samples probably occurs in the cytoplasm and/or other cytoplasmic organelles other than the mitochondrion. It is possible that the increase in adipose tissue in obesity enhances leptin production,31,54 with a decrease in adiponectin and, finally, a rise in insulin resistance.59 These present results indicate that melatonin may consequently enhance adiponectin production by raising Ca2+ levels in adipocytes. This increase in circulating adiponectin levels would be associated with a reduction in insulin resistance. The melatonin-induced increase in pancreas Ca2+ levels observed in our study may be related to the previously reported improvement in glucose uptake by skeletal muscle, liver and pancreas.45,62,63 In the same ZDF rats we also found that melatonin reduced fasting hyperglycemia by 18.6%, insulinemia by 15.9%, HOMA-IR by 31%, as well as free fatty acid levels by 13.5%.16 All these data demonstrated that oral melatonin administration ameliorated glucose homeostasis in ZDF rats. Similarly to our findings others also reported that melatonin treatment diminished insulin resistance, and reduced glucose levels and the HOMA index.18 The alteration of Ca2+ in pancreatic β-cells would impair insulin release in response to glucose, given observations of the association of insulin release with Ca2+ variations in pancreatic β-cells.64 Dou et al.65 recently showed that Ca2+ influx could activate a sustained insulin secretion in cultured pancreatic β-cells from rats. Hence, the increased Ca2+ in the pancreas observed in our study may be attributed to the fact that melatonin could modulate insulin release through calcium-related mechanisms, although the mechanism by which melatonin modifies calcium oscillations in pancreatic cells remains unknown. As shown in Fig. 1D and 3, brain and plasma Ca2+ levels were not affected by melatonin administration, suggesting that the increase in Ca2+ levels in the liver, muscle and adipose tissue of fatty rats chronically treated with melatonin is not directly related to previously elevated plasma Ca2+ levels. Nevertheless, we consider that blood must have served as a vehicle for Ca2+ to reach these organs. Moreover, the lack of changes in plasma Ca2+ levels with melatonin administration appears logical, given the close homeostatic regulation of Ca2+ at blood level due to its intervention in multiple important metabolic functions.39,66,67 The increase in tissues may be also attributable to the greater Ca2+ bioavailability in the diet.68 As previously mentioned, treatment of chicks with menadione impaired intestinal Ca2+ absorption through oxidative stress and apoptosis, and these alterations in calcium absorption were restored by the antioxidant actions of melatonin.68 Accordingly, blood would have served as a vehicle for the absorbed calcium, which was increased by the restoration of intestinal absorption due to the antioxidant action of melatonin on the increased oxidative stress in diabetes.69 It has also been reported that the impaired Ca2+ metabolism of Zücker diabetic fatty rats is related to an excessive urinary excretion of 1,25-dihydroxycholecalciferol, caused by the reducing absorption,70 however, the effects of melatonin on this hormone have not yet been elucidated.
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Animals and experimental protocols The study complied with the directives of the European Union and was approved by the ethics committee of the University of Granada. Sixty male Zücker rats were used in this study: 30 ZDF rats weighing 180–200 g, and 30 ZL rats weighing 120–140 g, obtained at the age of five weeks from the Charles River laboratory, Barcelona. Purina chow 5008 (58.5% carbohydrates, 6.5% fat, 23% protein, 4% fiber and 6.8% ashes) and tap water were available ad libitum.16,31 Food intake during the 6-week treatment period was around 2.5-fold higher in ZDF versus ZL groups. Animals were housed in transparent plastic cages (3–4 animals per cage) in a room with a 12 : 12 dark/light cycle (lights on at 07:00 h). At the age of 6 weeks, the ZDF and ZL groups were divided into three groups (n = 10 in each) to create control groups (ZDF-C and ZL-C), melatonin-treated groups (ZDF-M and ZL-M) and vehicle-treated groups (ZDF-V and ZL-V). Melatonin was administered in the drinking water at a dose of 10 mg per kg body weight per day diluted in a minimum volume of ethanol. In the ZDF-V and ZL-V groups the ethanol concentration was 0.066%. Melatonin and vehicle solutions were prepared every 2–3 days. At the end of the treatment period, the rats (12 weeks) were fasted overnight, anesthetized with thiobarbital sodium (thiopental) and sacrificed. Blood was drawn by cardiac puncture in the left ventricle, collected in ethylenediaminetetraacetic acid (EDTA) vacutainer tubes, centrifuged and divided into aliquots, freezing the plasma obtained at −80 °C. Samples of the liver, muscle, pancreas, brain, WAT fat pads (visceral omental, gonadal and renal) and lumbar subcutaneous fat were then gathered by surgical excision. The tissue samples were washed with a saline solution and stored at −80 °C until analysis. Equipment and reagents Ca2+ levels were measured using a 1100 B Atomic Absorption Spectrometer equipped with a Ca2+ and Mg2+ multi-element hollow cathode lamp (Perkin-Elmer, Germany). Reagent grade water was obtained using the R015 Milli-Q system (Waters, Medford, MA), and study tissue samples were mineralized in a Multiplaces Selecta mineralization block (Barcelona, Spain). Melatonin was obtained from Sigma Chemical (Madrid, Spain). Calibration lines were prepared with 1000 mg L−1 of Tritisol Ca2+ standard solution (Merck, Darmstadt, Germany). All solutions were prepared with analytical grade reagents: 65% HNO3, 60% HClO4, 37% HCl, 99.99% NaOH and La2O3 (Suprapur, Merck). Standard calibration solutions and dilutions were prepared with deionized bidistilled water (specific resistivity of 18 mΩ cm−1) obtained immediately before use by filtering distilled water through the Milli-Q purifier. Calcium level determination by flame atomic absorption spectrometry (AAS) Ca2+ levels in the tissues studied (liver, muscle, pancreas, brain, gonadal fat, renal visceral fat, white omental fat and
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lumbar subcutaneous fat) were determined by weighing 0.100–0.200 g of the samples in Pyrex glass tubes, adding 0.8 ml Nitric acid (HNO3), heating them at 80 °C for 15 min and then at 180 °C for 45 min. Next, 0.8 ml of a HNO3–HClO4 solution (4 : 1) was added and they were heated at 200 °C for 90 min. The resulting digested solution was then diluted to 2.5 ml with milli-Q water and maintained in this state until its analysis. The influence of the matrix on Ca2+ absorbance values was examined by applying the calibration addition method to the plasma and to each tissue. The calibration lines were obtained for each tissue by drawing 6 0.005 mL aliquots (same volume as analytical solution), placing them in 1 mL Eppendorf tubes and adding 0.100 mL of 1% Lanthanum chloride (LaCl3) to each aliquot along with increasing amounts of pattern Ca2+ solution (4 ppm). After dilution with bidistilled H2O to a final volume of 1 mL, the Ca2+ content was determined by flame AAS (wavelength of 422.7, slit width of 0.7 nm). The Ca2+ concentrations were added to each sample as a function of the concentrations already present. The final dilutions obtained were directly aspirated to the acetylene-air flame of the spectrophotometer, obtaining measurements of the absorbance at different Ca2+ concentrations in the samples. The accuracy and precision of the analytical procedure method was verified using a standard reference material with a certified Ca2+ content (Certified Reference Material Human Serum Chengdu ShuyangMedition Factory, Chengdu, China; National Research Center for CRM′1, Beijing, China; United Analysis and Measurement Center of Sichnan, Chengdu, China). No significant difference was found between the certified level and the result obtained (85.5 ± 5.3 mg L−1 vs. 84.6 ± 6.5 mg L−1, respectively, p > 0.05). Statistical analysis SPSS 17.0 (Chicago, IL) was used for data analyses. Results were expressed as the arithmetic mean ± standard error of the mean (SEM), the normality of the data distribution was verified with the Kolmogorov–Smirnov test and the homogeneity of variance with Levene’s test. Analysis of variance (ANOVA) and Duncan’s multiple range test were used to compare parametric variables and the Kruskal–Wallis test was applied to compare non-parametric variables. A two-tailed ANOVA was used to establish significant differences among subgroups. P < 0.05 was considered significant.
Conclusions In conclusion, chronic melatonin administrations at pharmacological doses to ZDF rats increased intracellular Ca2+ levels in the liver, muscle, pancreas and, more markedly, in gonadal fat, visceral renal fat, white omental fat and lumbar subcutaneous fat. Given the above data, we propose a possible additional action mechanism for the antidiabetic effect of melatonin related to its regulation of cellular Ca2+ homeostasis, opening up the possibility of using melatonin for the treat-
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ment of T2DM. Likewise, the increase in Ca2+ levels appears to enhance insulin sensitivity, which may in turn be related to the elevation of adiponectin levels after melatonin administration, with a subsequent increase in insulin signaling. Further research is warranted to establish the distribution of Ca2+ among the different cell compartments in these tissues.
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Ethical standards The experiments were approved by the Ethical Committee of the University of Granada (Granada, Spain) according to European Union guidelines.
Acknowledgements The study was supported by project SAF 2013–45752-R from the Ministerio de Economia y Competitividad (Spain), in part by the CTS-109 group from the Junta de Andalucía (Spain) and project FMHS/AA/Sd/26/13 from the United Arab Emirates University (UAEU) College of Medicine and Health Sciences. The authors thank Richard Davies for improving the English of the manuscript.
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10 A. I. Othman, M. A. El-Missiry, M. A. Amer and M. Arafa, Life Sci., 2008, 83, 563–568. 11 D. A. Castroviejo, L. C. López, G. Escames, A. López, J. A. García and R. J. Reiter, Curr. Top. Med. Chem., 2011, 11, 221–240. 12 A. Dey and J. Lakshmanan, Food Funct., 2013, 4, 1148–1184. 13 M. I. Rodríguez, G. Escames, L. C. López, A. López, J. A. García, F. Ortiz and D. Acuña, J. Pineal Res., 2007, 42, 272–279. 14 D. Bonnefont-Rousselot and F. Collin, Toxicology, 2010, 278, 55–67. 15 C. F. Chen, D. Wang, R. J. Reiter and D. Y. Yeh, J. Pineal Res., 2011, 50, 46–53. 16 A. Agil, I. Rosado, R. Ruiz, A. Figueroa, N. Zen and G. Fernandez-Vazquez, J. Pineal Res., 2012, 52, 203–210. 17 A. Agil, R. J. Reiter, A. Jimenez-Aranda, R. Iban-Arias, M. Navarro-Alarcon, J. A. Marchal, A. Adem and G. Fernandez-Vazquez, J. Pineal. Res., 2013, 54, 381–388. 18 J. A. Tresguerres, S. Cuesta, R. A. Kireev, C. Garcia, D. Acuna-Castroviejo and E. Vara, Horm. Mol. Biol. Clin. Investig., 2013, 16, 47–54. 19 J. Cipolla-Neto, F. G. Amaral, S. C. Afeche, D. X. Tan and R. J. Reiter, J. Pineal Res., 2014, 56, 371–381. 20 X. Cai, X. Wang and D. E. Clapham, Mol. Biol. Evol., 2014, 31, 2735–2740. 21 M. Shimodaira, T. Niwa, K. Nakajima, M. Kobayashi, N. Hanyu and T. Nakayama, Exp. Clin. Endocrinol. Diabetes, 2015, 123, 165–169. 22 S. Krauss, C. Y. Zhang and B. B. Lowell, Nat. Rev. Mol. Cell Biol., 2005, 6, 248–261. 23 H. P. Ammon, A. Fahmy, M. Mark, M. A. Wahl and N. Youssif, J. Physiol., 1987, 384, 347–354. 24 C. A. Stuart, D. Yin, M. E. Howell, R. J. Dykes, J. J. Laffan and A. A. Ferrando, Am. J. Physiol.: Endocrinol. Metab., 2006, 291, E1067–E1073. 25 S. Huang and M. P. Czech, Cell Metab., 2007, 5, 237–252. 26 W. W. Winder and D. G. Hardie, Am. J. Physiol., 1999, 277, E1–10. 27 O. B. Vadziuk, Ukr. Biokhim. Zh., 2014, 86, 5–22. 28 N. Jessen and L. J. Goodyear, J. Appl. Physiol., 2005, 99, 330–337. 29 A. J. Rose and E. A. Richter, Physiology, 2005, 20, 260–270. 30 Y. Mitani, A. Behrooz, G. R. Dubyak and F. Ismail-Beigi, Am. J. Physiol., 1995, 269, C1228–C1234. 31 A. Agil, M. Navarro-Alarcon, R. Ruiz, S. Abuhamadah, M. Y. El-Mir and G. F. Vazquez, J. Pineal. Res., 2011, 50, 207–212. 32 M. Navarro-Alarcon, F. J. Ruiz-Ojeda, R. M. BlancaHerrera, A. Kaki, A. Adem and A. Agil, Food Funct., 2014, 5, 512–516. 33 K. Fukunaga, K. Horikawa, S. Shibata, Y. Takeuchi and E. Miyamoto, J. Neurosci. Res., 2002, 70, 799–807. 34 I. Bazwinsky-Wutschke, E. Muhlbauer, E. Albrecht and E. Peschke, J. Pineal Res., 2014, 56, 439–449. 35 N. L. Harrison and M. Zatz, J. Neurosci., 1989, 9, 2462– 2467.
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36 C. S. Nelson, M. Ikeda, H. S. Gompf, M. L. Robinson, N. K. Fuchs, T. Yoshioka, K. A. Neve and C. N. Allen, Mol. Endocrinol., 2001, 15, 1306–1317. 37 S. A. Andrabi, I. Sayeed, D. Siemen, G. Wolf and T. F. Horn, FASEB J., 2004, 18, 869–871. 38 J. Pawlak, J. Singh, R. W. Lea and K. Skwarlo-Sonta, Mol. Cell. Biochem., 2005, 275, 207–213. 39 A. Guerrero-Hernandez and A. Verkhratsky, Cell Calcium, 2014, 56, 297–301. 40 K. W. Kinnally, P. M. Peixoto, S. Y. Ryu and L. M. Dejean, Biochim. Biophys. Acta, Mol. Cell Res., 2011, 1813, 616–622. 41 A. Jimenez-Aranda, G. Fernandez-Vazquez, A. S. M. Mohammad, R. J. Reiter and A. Agil, J. Pineal Res., 2014, 57, 103–109. 42 M. J. Tunon, B. San Miguel, I. Crespo, F. Jorquera, E. Santamaria, M. Alvarez, J. Prieto and J. GonzalezGallego, J. Pineal Res., 2011, 50, 38–45. 43 L. Yu, Y. Sun, L. Cheng, Z. Jin, Y. Yang, M. Zhai, H. Pei, X. Wang, H. Zhang, Q. Meng, Y. Zhang, S. Yu and W. Duan, J. Pineal Res., 2014, 57, 228–238. 44 X. Tan, X. Guo and H. Liu, Saudi Med. J., 2013, 34, 701–708. 45 M. J. Amaya and M. H. Nathanson, Compr. Physiol., 2013, 3, 515–539. 46 P. J. Bartlett, L. D. Gaspers, N. Pierobon and A. P. Thomas, Cell Calcium, 2014, 55, 306–316. 47 A. Agil, M. El-Hammadi, A. Jimenez-Aranda, M. Tassi, W. Abdo, G. Fernandez-Vazquez and R. J. Reiter, J. Pineal Res., 2015, DOI: 10.1111/jpi.12241. 48 D. E. James, R. Brown, J. Navarro and P. F. Pilch, Nature, 1988, 333, 183–185. 49 A. E. Stenbit, T. S. Tsao, J. Li, R. Burcelin, D. L. Geenen, S. M. Factor, K. Houseknecht, E. B. Katz and M. J. Charron, Nat. Med., 1997, 3, 1096–1101. 50 S. Lund, G. D. Holman, O. Schmitz and O. Pedersen, Proc. Natl. Acad. Sci. U. S. A., 1995, 92, 5817–5821. 51 H. Karaki, H. Ozaki, M. Hori, M. Mitsui-Saito, K. Amano, K. Harada, S. Miyamoto, H. Nakazawa, K. J. Won and K. Sato, Pharmacol. Rev., 1997, 49, 157–230. 52 B. Egan and J. R. Zierath, Cell Metab., 2013, 17, 162– 184. 53 R. S. Ahima, Obesity, 2006, 14(Suppl 5), 242S–249S. 54 B. L. Kasiske, M. P. O’Donnell and W. F. Keane, Hypertension, 1992, 19, I110–I115.
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Cite this: DOI: 10.1039/c5fo00590f
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Melatonin increases intracellular calcium in the liver, muscle, white adipose tissues and pancreas of diabetic obese rats A. Agil,*a E. K. Elmahallawy,a J. M. Rodríguez-Ferrer,b A. Adem,c S. M. Bastaki,c I. Al-abbadi,d Y. A. Fino Solanoa,e and M. Navarro-Alarcón*e Melatonin, a widespread substance with antioxidant and anti-inflammatory properties, has been found to act as an antidiabetic agent in animal models, regulating the release and action of insulin. However, the molecular bases of this antidiabetic action are unknown, limiting its application in humans. Several studies have recently shown that melatonin can modify calcium (Ca2+) in diabetic animals, and Ca2+ has been reported to be involved in glucose homeostasis. The objective of the present study was to assess whether the antidiabetic effect of chronic melatonin at pharmacological doses is established via Ca2+ regulation in different tissues in an animal model of obesity-related type 2 diabetes, using Zücker diabetic fatty (ZDF) rats and their lean littermates, Zücker lean (ZL) rats. After the treatments, flame atomic absorption spectrometry was used to determine Ca2+ levels in the liver, muscle, main types of internal white adipose tissue, subcutaneous lumbar fat, pancreas, brain, and plasma. This study reports for the first time that chronic melatonin administration (10 mg per kg body weight per day for 6 weeks) increases Ca2+ levels in muscle, liver, different adipose tissues, and pancreas in ZDF rats, although there were no significant
Received 22nd May 2015, Accepted 23rd June 2015
changes in their brain or plasma Ca2+ levels. We propose that this additional peripheral dual action
DOI: 10.1039/c5fo00590f
mechanism underlies the improvement in insulin sensitivity and secretion previously documented in samples from the same animals. According to these results, indoleamine may be a potential candidate for
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the treatment of type 2 diabetes mellitus associated with obesity.
Introduction Melatonin, N-acetyl-5-methoxytryptamine, is an indoleamine mainly produced in peripheral organs and tissues in larger amounts than in the pineal gland, although pineal, but not extrapineal melatonin, retains chronobiotic properties.1–5 The widespread presence of melatonin and its variety of biological properties suggest that it is a very ancient molecule with important roles in phylogenetic distant organisms.6 Various researchers have described this indolamine as a highly interesting option for maintaining optimal health conditions.2,7–9 Melatonin has been found to exert an antioxidant effect via
a Department of Pharmacology and Neurosciences Institute (CIBM), School of Medicine, University of Granada, Spain. E-mail: [email protected]; Fax: +34958243537 b Department of Physiology and Neurosciences Institute (CIBM), School of Medicine, University of Granada, Spain c Department of Pharmacology, School of Medicine and Health Sciences, University of United Arab Emirates, Al Ain, United Arab Emirates d 1678 School of Pharmacy, University of Jordan, Amman, Jordan e Department of Nutrition and Food Science, School of Pharmacy, University of Granada, Spain
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different mechanisms,7,8,10–12 confirming its role in the control of inflammation and oxidative stress, which underlie various chronic diseases.13–18 Some authors have proposed that melatonin may be useful against the increasing pandemic of overweight, obesity and obesity-related diabetes worldwide.19 These reports are in the same line as previous observations by our group that chronic melatonin administration improves diabetes by ameliorating the homeostatic model assessment insulin resistance (HOMA-IR) and homeostasis model assessment β-cell functionality (HOMA-IH),16 and counteracts oxidative stress and low-grade inflammation in the ZDF diabetes/obesity model.16 Furthermore, several studies have demonstrated that age-related insulin secretion dysfunction plays an important role in glucose metabolism disorders at older ages, contributing to the high indexes of glucose intolerance in elderly populations.17,18 Calcium (Ca2+) is a controlling messenger in eukaryotic cell signaling and is involved in a plethora of events in different biological models.20 Ca2+ was found to play a key role in β-cell functionality,21 and a lower intracellular level of Ca2+ was associated with the presence of hyperglycemia and a depolarizing K+ stimulus.22 These effects may also be related to a poss-
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ible alteration of the adenosine triphosphate (ATP)-sensitive K+ channel (KATP channel). Hence, a decrease in Ca2+ regulation and glucose signaling appears to contribute to the reduction in β-cell stimulus-insulin secretion coupling in rats of an advanced age.23 Additionally, HOMA-IR studies in key tissues revealed a relationship between insulin tolerance and adequate Ca2+ levels in muscle cells.22 Glucose transporter type 4 (GLUT4) is known to be highly expressed in adipose tissue, skeletal, muscle and liver.24,25 Two cellular events that contribute to the improvement of GLUT4 translocation to the cell surface have been observed during muscle contraction: a transient increase in intracellular Ca2+ concentration ([Ca2+]i) and elevation of the [ATP]/[AMP] ratio.25–27 This process is mediated by the activation of the Ca2+/calmodulin-dependent protein kinase (CaMKII), which is also required for Ca2+ homeostasis and its reuptake.28,29 It has also been reported that a calcium ionophore-induced rise in cytosolic calcium produces an increase in glucose transporter type 1 (GLUT1) protein and mRNA, which enhances the entry of glucose.6,30 We have found no published study on the effect of melatonin supplementation on the regulation of Ca2+ levels in tissues. Our group previously reported that melatonin administrations improve various biomarkers associated with type 2 diabetes mellitus (T2DM) through its effect on chromium (Cr) and vanadium (V).16,17,31,32 Importantly, melatonin also affects CaMKII protein levels, probably acting indirectly via the Ca2+calmodulin signaling pathway for regulating insulin secretion.33 Hence, melatonin can enhance the capacity of β cells to respond to metabolic signals by impacting on this kinase group.34 With this background, the objective of the present study was to investigate the effects of chronic melatonin administration on Ca2+ homeostasis in liver, muscle, the main types of internal white adipose tissues (visceral omentum, gonadal and renal), subcutaneous lumbar fat, pancreas, brain, pancreas and plasma in young male ZDF rats and their lean littermates (ZL rats).
Results and discussion As shown in Fig. 1–3, the four subgroups significantly differed (P < 0.05) in Ca2+ levels measured in the liver, muscle, pancreas, WAT [visceral omental, gonadal and renal] and lumbar subcutaneous fat samples but not in those measured in brain or plasma samples (P > 0.05). The ZDF-M group showed a significantly higher mean Ca2+ level in: liver versus ZDF-C, ZL-C and ZL-M groups (0.185 ± 0.031 vs. 0.073 ± 0.023, 0.115 ± 0.024 and 0.070 ± 0.040 mg g−1, respectively; P < 0.05; Fig. 1A); muscle versus ZDF-C and ZL-C groups (0.248 ± 0.033 vs. 0.095 ± 0.033 and 0.131 ± 0.022 mg g−1, respectively; P < 0.05; Fig. 1B); and gonadal fat versus the ZDF-C, ZL-C and ZL-M groups (0.876 ± 0.217 vs. 0.239 ± 0.123, 0.042 ± 0.015 and 0.052 ± 0.019 mg g−1, respectively, P < 0.01). Higher mean Ca2+ levels were found in the omental white fat of the ZDF-M and ZL-M
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groups versus the ZDF-C group (0.201 ± 0.020 and 0.160 ± 0.015 vs. 0.081 ± 0.015 mg g−1, respectively, P < 0.05 Fig. 2A and B), and the ZDF-M group showed a significantly higher mean Ca2+ level versus the ZDF-C group in renal visceral WAT (from 0.064 ± 0.040 to 0.201 ± 0.026 mg g−1, P < 0.05; Fig. 2C), lumbar subcutaneous fat (from 0.093 ± 0.068 to 0.223 ± 0.055 mg g−1, P < 0.01 Fig. 2D) and pancreas (from 0.194 ± 0.091 to 0.336 ± 0.030 mg g−1, P < 0.05; Fig. 1C). No significant differences in brain (Fig. 1D) or plasma (Fig. 3) Ca2+ levels were found among the four study subgroups (P > 0.05). No significant differences were found in the mean Ca2+ levels of any tissue (including plasma) between the ZDF-C and ZL-C groups, as exhibited in the figures, or between the control and vehicle-treated animals (data not shown). In this study, the chronic administration of melatonin during 6 weeks in diabetic obese ZDF rats at a dose within the pharmacological range (10 mg per kg body weight per day) increased their intracellular Ca2+ levels in the liver, muscle and pancreas but had no effect on their brain tissue or plasma Ca2+ concentrations in ZDF rats. These findings add to evidence previously obtained in samples from the same animal on the positive effects of melatonin on fasting glucose and insulin blood levels (β-cell function, as estimated by HOMA1%B index), insulin resistance (HOMA-IR index), and the leptin/ adiponectin ratio.16,31 These findings together with the great role played by melatonin in the reduction of free fatty acid and oxidative or nitrosative stress emphasize the beneficial effect of melatonin treatment on age related insulin resistance and on the development of T2DM.18 As noted above, melatonin administration can increase plasma levels of V and Cr, which are both related to glucose tolerance,32 and it can reduce the body weight of ZDF rats for the same food intake, related to an improved lipid profile.31 The relationship between melatonin and Ca2+ flux regulation has been studied in numerous cells.35–37 In pancreatic β cells, hyperglycemia and hyperlipidemia were reported to reduce insulin sensitivity by hindering Ca2+ entry into the cell due to the high [AMP]/[ATP] ratio.22 Other researchers found that melatonin increased intracellular Ca2+ in macrophages of streptozotocin-induced diabetic rats.38 In addition, the hyperglycemic and hyperlipidemic characteristics of T2DM in proopiomelanocortin (POMC) system cells and pancreatic β cells have been associated with an increase in superoxide radicals and therefore in the expression of protein uncoupling protein2 (UCP2). This would reduce ATP production and consequently the ATP/ADP ratio.22 Hence, there is a reduced expression in fatty rats of the ATP-dependent K+ channel (KATP channel) component, designated Kir6.2 protein (Potassium Channel, Inwardly Rectifying, Kir6.2), which hinders K+ channel closure.33 As a result, the voltage-sensitive calcium channel (VSCC) system is not blocked, ultimately impeding Ca2+ entry into the cell, which facilitates insulin sensitivity loss and exacerbates T2DM. It should be remembered that the ADP/ATP ratio can be reduced by increased ATP synthesis, favoring the closure of the KATP kir6.2 channel, opening the cell membrane Ca2+ channel, increasing intracellular Ca2+, improving
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Fig. 1 (A) Effect of melatonin treatment on calcium levels (mg g−1) in the liver. Values are means ± SEM ZDF, Zücker Diabetic Fatty rats; ZL, Zücker Lean rats. *P < 0.05, **P < 0.01. (B) Effect of melatonin treatment on calcium levels (mg g−1) in skeletal muscle. Values are means ± SEM ZDF, Zücker Diabetic Fatty rats; ZL, Zücker Lean rats. ***P < 0.001. (C) Effect of melatonin treatment on calcium levels (mg g−1) in the pancreas. Values are means ± SEM ZDF, Zücker Diabetic Fatty rats; ZL, Zücker Lean rats. **P < 0.01. (D) Effect of melatonin treatment on calcium levels (mg g−1) in the brain. Values are means ± SEM ZDF, Zücker Diabetic Fatty rats; ZL, Zücker Lean rats.
glucose signaling and reducing insulin resistance, while calcium signaling in mitochondria stimulates ATP production.22,39 The major role played by melatonin at the subcellular level can be largely attributed to its dual effect on mitochondrial permeability transition pore (mPTP) opening, the gatekeeper of apoptotic and necrotic cell death, which consequently controls Ca2+ homeostasis.37,40 In this line, it was recently observed that chronic melatonin administrations markedly inhibit Ca2+-induced mPTP opening in mitochondria isolated from white and beige fat of both diabetic fatty and lean rats.41 Melatonin may contribute via these mechanisms to decrease oxidative stress and therefore reduce the induction of
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mPTP opening, maintaining intramitochondrial Ca2+ levels. However, the increased Ca2+ in the studied tissues, especially in WAT [visceral omental, gonadal and renal] and lumbar subcutaneous fat samples, does not imply an increase in mitochondrial levels, because melatonin was found to inhibit Ca2+induced mPTP opening in isolated mitochondria,41 but it is associated with the decreased oxidative stress described in previous studies using the same animal model.17,41 It should be taken into account that the calcium-activation of mPTP is inhibited by the overexpression of antiapoptotic B-cell lymphoma 2 (bcl-2),40 which is induced by melatonin treatment in liver,42 heart43 and brain,44 among other tissues.
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Fig. 2 (A) Effect of melatonin treatment on calcium levels (mg g−1) in omental white adipose tissue. Values are means ± SEM ZDF, Zücker Diabetic Fatty rats; ZL, Zücker Lean rats. *P < 0.05, **P < 0.01. (B) Effect of melatonin treatment on calcium levels (mg g−1) in gonadal white adipose tissue. Values are means ± SEM ZDF, Zücker Diabetic Fatty rats; ZL, Zücker Lean rats. *P < 0.05, ***P < 0.001. (C) Effect of melatonin treatment on calcium levels (mg g−1) in renal white adipose tissue. Values are means ± SEM ZDF, Zücker Diabetic Fatty rats; ZL, Zücker Lean rats. *P < 0.05. (D) Effect of melatonin treatment on calcium levels (mg g−1) in subcutaneous fat. Values are means ± SEM ZDF, Zücker Diabetic Fatty rats; ZL, Zücker Lean rats. **P < 0.01.
The liver is considered the main organ involved in maintaining normal glucose homeostasis through its role in regulating hepatic gluconeogenesis and glycolysis.45,46 As depicted in Fig. (1A), liver Ca2+ levels were higher in melatonin-treated ZDF rats than in the other groups (ZDF-C, ZL-M and ZL-C), which may also be related to an improved glucose tolerance, in common with previous studies which showed that melatonin is very useful in the protection of steatotic livers in ZDF rats which is not found in ZL rats.46,47 The higher levels of Ca2+ in melatonin-treated ZDF rats are consistent with various reports of its association with a decrease in insulin resistance.22,31,32 In humans, GLUT4
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protein is encoded by the GLUT4 gene and is a well-known insulin-regulated glucose transporter.24,25 It is highly expressed in adipose tissues and striated muscle and is closely related to glucose levels.48–50 Muscle contraction transiently increases the intracellular cytoplasmic concentration of Ca2+ with a concomitant elevation of the AMP/ATP ratio,51,52 which improves the glucose uptake of GLUT4 and glucose tolerance observed in this animal model of obesity and diabetes.31 Therefore, melatonin might indirectly enhance the externalization of the GLUT-4 receptor, improving glucose homeostasis in the tissues in which Ca2+ levels were increased. Contrarily, it has been lately reported that melatonin reduces the uptake of
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Fig. 3 Effect of melatonin treatment on calcium levels (mg ml−1) in plasma. Values are means ± SEM ZDF, Zücker Diabetic Fatty rats; ZL, Zücker Lean rats.
glucose and modifies the expression of GLUT1 transported in prostate cancer cells.6 Melatonin-treated ZDF rats also showed significantly higher Ca2+ levels in comparison to untreated ZDF-C rats in all adipose tissue samples (WAT [visceral omental, gonadal and renal] and lumbar subcutaneous fat). In obesity-related T2DM, adipose tissue was reported to actively participate in metabolic homeostasis through its capacity to produce adipocytokines, including leptin and adiponectin.31,53 Leptin is produced by mature adipocytes that have been hypertrophied due to fat accumulation. Most studies have observed elevated blood leptin levels (hyperleptinemia) in ZDF rats, attributable to their strong genetic resistance to this adipocytokine through the homozygous mutation of its receptor, which therefore has virtually no signaling capacity. This situation produces hyperphagia and reduced energy expenditure, resulting in severe obesity.54 Various authors have observed a reduction in leptin levels after melatonin administration.55–58 It has also been reported that melatonin-induced weight loss may improve the adipocyte metabolism and thereby increase the secretion of adiponectin, whose levels are significantly reduced in fatty animals.31 This finding reported by our research group was in samples from the same ZDF rats where melatonin raised hypoadiponectinemia by 40% and concomitantly decreased hyperleptinemia by 34%.16 Circulating adiponectin levels are generally inversely correlated with fatty mass and insulin resistance, being lower in obesity and T2DM in both animals and humans.59 This is of special relevance, given that adiponectin is an insulin sensitizer60 in the liver, muscle and peripheral tissues,61 and low levels increase the risk of T2DM.60 Adiponectin modulation may be a potential therapeutic strategy by using certain Ca2+ channel blockers to enhance its levels, which may help to suppress or prevent the onset of diseases related to inflammatory processes, such as atherogenesis.60 In the present study, melatonin administration increased the levels of Ca2+ in all studied adipose tissues (white omental, gonadal, renal and lumbar subcutaneous fat),
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and its metabolism is considerably altered in obesity and diabetes. The Ca2+ increase observed in the present adipose tissue samples probably occurs in the cytoplasm and/or other cytoplasmic organelles other than the mitochondrion. It is possible that the increase in adipose tissue in obesity enhances leptin production,31,54 with a decrease in adiponectin and, finally, a rise in insulin resistance.59 These present results indicate that melatonin may consequently enhance adiponectin production by raising Ca2+ levels in adipocytes. This increase in circulating adiponectin levels would be associated with a reduction in insulin resistance. The melatonin-induced increase in pancreas Ca2+ levels observed in our study may be related to the previously reported improvement in glucose uptake by skeletal muscle, liver and pancreas.45,62,63 In the same ZDF rats we also found that melatonin reduced fasting hyperglycemia by 18.6%, insulinemia by 15.9%, HOMA-IR by 31%, as well as free fatty acid levels by 13.5%.16 All these data demonstrated that oral melatonin administration ameliorated glucose homeostasis in ZDF rats. Similarly to our findings others also reported that melatonin treatment diminished insulin resistance, and reduced glucose levels and the HOMA index.18 The alteration of Ca2+ in pancreatic β-cells would impair insulin release in response to glucose, given observations of the association of insulin release with Ca2+ variations in pancreatic β-cells.64 Dou et al.65 recently showed that Ca2+ influx could activate a sustained insulin secretion in cultured pancreatic β-cells from rats. Hence, the increased Ca2+ in the pancreas observed in our study may be attributed to the fact that melatonin could modulate insulin release through calcium-related mechanisms, although the mechanism by which melatonin modifies calcium oscillations in pancreatic cells remains unknown. As shown in Fig. 1D and 3, brain and plasma Ca2+ levels were not affected by melatonin administration, suggesting that the increase in Ca2+ levels in the liver, muscle and adipose tissue of fatty rats chronically treated with melatonin is not directly related to previously elevated plasma Ca2+ levels. Nevertheless, we consider that blood must have served as a vehicle for Ca2+ to reach these organs. Moreover, the lack of changes in plasma Ca2+ levels with melatonin administration appears logical, given the close homeostatic regulation of Ca2+ at blood level due to its intervention in multiple important metabolic functions.39,66,67 The increase in tissues may be also attributable to the greater Ca2+ bioavailability in the diet.68 As previously mentioned, treatment of chicks with menadione impaired intestinal Ca2+ absorption through oxidative stress and apoptosis, and these alterations in calcium absorption were restored by the antioxidant actions of melatonin.68 Accordingly, blood would have served as a vehicle for the absorbed calcium, which was increased by the restoration of intestinal absorption due to the antioxidant action of melatonin on the increased oxidative stress in diabetes.69 It has also been reported that the impaired Ca2+ metabolism of Zücker diabetic fatty rats is related to an excessive urinary excretion of 1,25-dihydroxycholecalciferol, caused by the reducing absorption,70 however, the effects of melatonin on this hormone have not yet been elucidated.
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Animals and experimental protocols The study complied with the directives of the European Union and was approved by the ethics committee of the University of Granada. Sixty male Zücker rats were used in this study: 30 ZDF rats weighing 180–200 g, and 30 ZL rats weighing 120–140 g, obtained at the age of five weeks from the Charles River laboratory, Barcelona. Purina chow 5008 (58.5% carbohydrates, 6.5% fat, 23% protein, 4% fiber and 6.8% ashes) and tap water were available ad libitum.16,31 Food intake during the 6-week treatment period was around 2.5-fold higher in ZDF versus ZL groups. Animals were housed in transparent plastic cages (3–4 animals per cage) in a room with a 12 : 12 dark/light cycle (lights on at 07:00 h). At the age of 6 weeks, the ZDF and ZL groups were divided into three groups (n = 10 in each) to create control groups (ZDF-C and ZL-C), melatonin-treated groups (ZDF-M and ZL-M) and vehicle-treated groups (ZDF-V and ZL-V). Melatonin was administered in the drinking water at a dose of 10 mg per kg body weight per day diluted in a minimum volume of ethanol. In the ZDF-V and ZL-V groups the ethanol concentration was 0.066%. Melatonin and vehicle solutions were prepared every 2–3 days. At the end of the treatment period, the rats (12 weeks) were fasted overnight, anesthetized with thiobarbital sodium (thiopental) and sacrificed. Blood was drawn by cardiac puncture in the left ventricle, collected in ethylenediaminetetraacetic acid (EDTA) vacutainer tubes, centrifuged and divided into aliquots, freezing the plasma obtained at −80 °C. Samples of the liver, muscle, pancreas, brain, WAT fat pads (visceral omental, gonadal and renal) and lumbar subcutaneous fat were then gathered by surgical excision. The tissue samples were washed with a saline solution and stored at −80 °C until analysis. Equipment and reagents Ca2+ levels were measured using a 1100 B Atomic Absorption Spectrometer equipped with a Ca2+ and Mg2+ multi-element hollow cathode lamp (Perkin-Elmer, Germany). Reagent grade water was obtained using the R015 Milli-Q system (Waters, Medford, MA), and study tissue samples were mineralized in a Multiplaces Selecta mineralization block (Barcelona, Spain). Melatonin was obtained from Sigma Chemical (Madrid, Spain). Calibration lines were prepared with 1000 mg L−1 of Tritisol Ca2+ standard solution (Merck, Darmstadt, Germany). All solutions were prepared with analytical grade reagents: 65% HNO3, 60% HClO4, 37% HCl, 99.99% NaOH and La2O3 (Suprapur, Merck). Standard calibration solutions and dilutions were prepared with deionized bidistilled water (specific resistivity of 18 mΩ cm−1) obtained immediately before use by filtering distilled water through the Milli-Q purifier. Calcium level determination by flame atomic absorption spectrometry (AAS) Ca2+ levels in the tissues studied (liver, muscle, pancreas, brain, gonadal fat, renal visceral fat, white omental fat and
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lumbar subcutaneous fat) were determined by weighing 0.100–0.200 g of the samples in Pyrex glass tubes, adding 0.8 ml Nitric acid (HNO3), heating them at 80 °C for 15 min and then at 180 °C for 45 min. Next, 0.8 ml of a HNO3–HClO4 solution (4 : 1) was added and they were heated at 200 °C for 90 min. The resulting digested solution was then diluted to 2.5 ml with milli-Q water and maintained in this state until its analysis. The influence of the matrix on Ca2+ absorbance values was examined by applying the calibration addition method to the plasma and to each tissue. The calibration lines were obtained for each tissue by drawing 6 0.005 mL aliquots (same volume as analytical solution), placing them in 1 mL Eppendorf tubes and adding 0.100 mL of 1% Lanthanum chloride (LaCl3) to each aliquot along with increasing amounts of pattern Ca2+ solution (4 ppm). After dilution with bidistilled H2O to a final volume of 1 mL, the Ca2+ content was determined by flame AAS (wavelength of 422.7, slit width of 0.7 nm). The Ca2+ concentrations were added to each sample as a function of the concentrations already present. The final dilutions obtained were directly aspirated to the acetylene-air flame of the spectrophotometer, obtaining measurements of the absorbance at different Ca2+ concentrations in the samples. The accuracy and precision of the analytical procedure method was verified using a standard reference material with a certified Ca2+ content (Certified Reference Material Human Serum Chengdu ShuyangMedition Factory, Chengdu, China; National Research Center for CRM′1, Beijing, China; United Analysis and Measurement Center of Sichnan, Chengdu, China). No significant difference was found between the certified level and the result obtained (85.5 ± 5.3 mg L−1 vs. 84.6 ± 6.5 mg L−1, respectively, p > 0.05). Statistical analysis SPSS 17.0 (Chicago, IL) was used for data analyses. Results were expressed as the arithmetic mean ± standard error of the mean (SEM), the normality of the data distribution was verified with the Kolmogorov–Smirnov test and the homogeneity of variance with Levene’s test. Analysis of variance (ANOVA) and Duncan’s multiple range test were used to compare parametric variables and the Kruskal–Wallis test was applied to compare non-parametric variables. A two-tailed ANOVA was used to establish significant differences among subgroups. P < 0.05 was considered significant.
Conclusions In conclusion, chronic melatonin administrations at pharmacological doses to ZDF rats increased intracellular Ca2+ levels in the liver, muscle, pancreas and, more markedly, in gonadal fat, visceral renal fat, white omental fat and lumbar subcutaneous fat. Given the above data, we propose a possible additional action mechanism for the antidiabetic effect of melatonin related to its regulation of cellular Ca2+ homeostasis, opening up the possibility of using melatonin for the treat-
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ment of T2DM. Likewise, the increase in Ca2+ levels appears to enhance insulin sensitivity, which may in turn be related to the elevation of adiponectin levels after melatonin administration, with a subsequent increase in insulin signaling. Further research is warranted to establish the distribution of Ca2+ among the different cell compartments in these tissues.
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Ethical standards The experiments were approved by the Ethical Committee of the University of Granada (Granada, Spain) according to European Union guidelines.
Acknowledgements The study was supported by project SAF 2013–45752-R from the Ministerio de Economia y Competitividad (Spain), in part by the CTS-109 group from the Junta de Andalucía (Spain) and project FMHS/AA/Sd/26/13 from the United Arab Emirates University (UAEU) College of Medicine and Health Sciences. The authors thank Richard Davies for improving the English of the manuscript.
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