Endocrine https://doi.org/10.1007/s12020-018-1653-x
ORIGINAL ARTICLE
Melatonin treatment suppresses appetite genes and improves adipose tissue plasticity in diet-induced obese zebrafish G. Montalbano 1,2 M. Mania1 F. Abbate1,2 M. Navarra3 M. C. Guerrera1,2 R. Laura1 J. A. Vega4,5 M. Levanti1,2 A. Germanà1,2 ●
●
●
●
●
●
●
●
1234567890();,:
1234567890();,:
Received: 16 February 2018 / Accepted: 12 June 2018 © Springer Science+Business Media, LLC, part of Springer Nature 2018
Abstract Purpose Overweight and obesity are important risk factors for diabetes, cardiovascular diseases, and premature death in modern society. Recently, numerous natural and synthetic compounds have been tested in diet-induced obese animal models, to counteract obesity. Melatonin is a circadian hormone, produced by pineal gland and extra-pineal sources, involved in processes which have in common a rhythmic expression. In teleost, it can control energy balance by activating or inhibiting appetite-related peptides. The study aims at testing effects of melatonin administration to control-fed and overfed zebrafish, in terms of expression levels of orexigenic (Ghrelin, orexin, NPY) and anorexigenic (leptin, POMC) genes expression and morphometry of visceral and subcutaneous fat depots. Methods Adult male zebrafish (n = 56) were divided into four dietary groups: control, overfed, control + melatonin, overfed + melatonin. The treatment lasted 5 weeks and BMI levels of every fish were measured each week. After this period fishes were sacrificed; morphological and morphometric studies have been carried out on histological sections of adipose tissue and adipocytes. Moreover, whole zebrafish brain and intestine were used for qRT-PCR. Results Our results demonstrate that melatonin supplementation may have an effect in mobilizing fat stores, in increasing basal metabolism and thus in preventing further excess fat accumulation. Melatonin stimulates the anorexigenic and inhibit the orexigenic signals. Conclusions It seems that adequate melatonin treatment exerts anti-obesity protective effects, also in a diet-induced obesity zebrafish model, that might be the result of the restoration of many factors: the final endpoint reached is weight loss and stabilization of weight gain. Keywords Melatonin Diet-induced obesity Zebrafish Adipose tissue.These authors contributed equally: Montalbano G. and Mania ●
●
●
M.
Introduction * G. Montalbano
[email protected] 1
Department of Veterinary Sciences, University of Messina, Polo Universitario SS. Annunziata, Messina 98168, Italy
2
Zebrafish Neuromorphology Lab, University of Messina, Polo Universitario SS. Annunziata, Messina 98168, Italy
3
Department of Drug Sciences and products for Health, University of Messina, Polo Universitario SS. Annunziata, Messina 98168, Italy
4
Departamento de Morfología y Biología Celular, Universidad de Oviedo, Oviedo, España 33006, Spain
5
Facultad de Ciencias de la Salud, 5 Poniente 1670, Universidad Autónoma de Chile, Talca, Chile
Obesity and its comorbidities represent the fifth leading cause of mortality worldwide since they are important risk factors for diabetes, cardiovascular diseases, cancer, and premature death [1] and the number of overweight or obese people is exponentially increasing [2, 3]. The causes of the increase in fat mass must be exogenous and endogenous factors [4], relying on a complex neuroanatomical network between brain and peripheral signals, controlling information on the balance between the calorie intake and the energy expenditure [5]. In this way, numerous synthetic and natural compounds that interact within this network have been studied to counteract obesity. Melatonin plays a significant role in the vertebrate circadian clock as a neuroendocrine hormone, and it is related to many regulatory
Endocrine
processes which have in common a rhythmic expression [6] such as food intake and metabolism [7], adiposity and seasonal body weight variation [8], glucose uptake [9], or intestinal reflexes [10]. Melatonin seems to play also a role in the regulation of energy homeostasis and food intake [11]. It is secreted and released mainly by pineal gland with a circadian pattern [12] and it is ubiquitous among living beings, such as plants [13], microbes, animals, humans; extra-pineal sources also produce it, independently of the photoperiod [13–15]. Particularly in the gastrointestinal tract of mammals, birds, and fish [16–18], melatonin is produced by enterochromaffin cells [19, 20] and it is believed to act as a paracrine hormone which can be secreted in both a continuous or a cyclic fashion. In teleost fish, melatonin can reduce food intake affecting protein, carbohydrate, and lipid metabolism [21– 23], and control energy balance by activating orexigenic and inhibiting anorexigenic peptides [20, 24]. In such context, melatonin has emerged as a candidate to control obesity [25]. Furthermore, convincing evidence exists for an association of circadian system derangement (chronodisruption) with sleep deprivation and melatonin suppression in obesity [24, 26]. Nevertheless, the real role of melatonin in the regulation of food intake and energy balance remains to be fully elucidated. Zebrafish is a common model used in biomedical research including metabolic diseases like obesity [27]. Recently, researchers have developed a zebrafish model that can successfully mimic diet-induced obesity [28, 29]. Overfed zebrafish accumulate white fat in the same deposits as humans [30, 31]. Therefore, to date many questions remain open and challenging, about a putative role of melatonin in ameliorating metabolic syndrome and associated comorbidities. For these reasons we planned an experimental protocol to examine in depth the effects of exogenous melatonin treatment in normal and diet-induced obese zebrafish, to further elucidate molecular and physiological mechanisms on which melatonin acts to improve obese phenotype. This paper could contribute to widen actual knowledge on potential use of melatonin as a putative natural treatment for obesity and obesity-related disorders.
Materials and methods Zebrafish husbandry and melatonin treatment In this study we used adult (3–9 month old) male zebrafish (n = 56) obtained from our breeding colony from C.I.S.S. (Centre of Experimental Ichthyopathology of Sicily, Department of Veterinary Science, University of Messina—
Italy), kept at constant temperature of 28.5 °C and fed once a day. The fish used for the experiment were maintained with a natural photoperiod (14 h light / 10 h dark) and divided into four dietary groups (n = 14 fish per group) as follows: the first maintenance group—used as control—was fed with the equivalent of 20 mg cysts/fish/day of freshly hatched Artemia nauplii (control, ctrl); the second group was fed with the equivalent of 60 mg cysts/fish/day (20 mg cysts/fish, three times a day) of freshly hatched Artemia nauplii (overfed, OF); the third group received the equivalent of 20 mg cysts/fish/day of freshly hatched Artemia nauplii and melatonin in water (normal feeding with melatonin, ctrl + MEL); the fourth group received the equivalent of 60 mg cysts/fish/day of freshly hatched Artemia nauplii (20 mg cysts/fish, three times a day) and melatonin in water (overfed with melatonin treatment, OF + MEL). The entire protocol took 5 weeks for completion. Zebrafish were separated in different tanks (1 zebrafish per 1-l tank), to ensure that the amount of food and melatonin provided would have been used by a single fish. Water was totally changed every 24 h to avoid melatonin accumulation in water, according to the protocol set up by Piccinetti et al. [20]. Melatonin was administered every day before feeding fish as follows: a stock solution (1 mM) was prepared by dissolving melatonin powder (Sigma-Aldrich, Saint Louis, MO, USA) in ethanol (50 mg/mL) and then in distilled water; 1 ml of this solution was dissolved in each fish tank of the third and fourth group (n = 28 fish in total to be treated with melatonin), in order to obtain the working solution (1 μM) in the fish tank itself.
Body mass index (BMI) measurements and tissue treatment During the treatment, to analyze the increase in body weight and BMI levels, the length and the body weight of every fish were measured each week. BMI was calculated by dividing body weight (g) by the square of body length (cm2), from the tip of the mouth to the end of the body. Then, BMI measurements were normalized by dividing each BMI value by the first value calculated for each group at the beginning of the experimental period. After a 5-week treatment, the fish were fasted for 24 h and then anaesthetized with MS222 (ethyl 3-aminobenzoate methanesulfonate, 0.2 g/l; Sigma, Saint Louis, MO, USA). Successively, heads and bodies of the animals were fixed for 24 h in Bouin’s fixative and routinely processed for light microscopy and morphometry (n = 5 fish per group), while brains and intestines of the remaining animals were pooled (n = 3 samples per pool, three pools in total) in RNA stabilization solution (RNA later, Ambion Life Technology) and quickly processed for the isolation of RNA and qRTPCR (n = 9 fish per group).
Endocrine
qRT-PCR: mRNA expression of obesity genes
Statistical analysis
The procedure used for qRT-PCR were carried out according to the procedure by Levanti et al. [32]. 1 μg of the cDNA was used to detect leptin, orexin, ghrelin, NPY (Neuropeptide Y) and POMC (Pro-opiomelanocortin) expression using qRT-PCR. The primers used were designed on the mRNA sequences published for leptin, ghrelin, NPY, orexin, POMC and β-actin (GenBank accession numbers: leptin a NM_001128576, ghrelin EU908735.1, NPY BC162071, orexin NM_001077392.2, POMC AY125332.2 and β-actin NM_131031) as follows: Leptin (GenBank accession no. NM_001128576): forward 5′-CATCATCGTCAGAATCAGGG-3′; reverse 5′ATCTCGGCGTATCTGGTCAA-3′; Ghrelin (GenBank accession no. EU908735.1): forward 5′-CAAGAGTGGGCAGAAGAGAA-3′; reverse 5′CTGAAGCACGGGACCATATT-3′; Orexin (GenBank accession no. NM_001077392.2): forward 5′-GCTCCTTGCAAACTACGAG-3′; reverse 5′GAGTTGTGCAGCAGCAGTTG-3′; POMC (GenBank accession no. AY125332.2): forward 5′-TGAACAGATAGAGCCGGAGT-3′; reverse 5′ACCTCGTTATTTGCCAGTC-3′; NPY (GenBank accession no. BC162071): forward 5′TGAAGATGTGGATGAGCTGG-3′; reverse 5′-CACCATGCCAAATGATCCTC-3′; β-actin (GenBank accession no. NM_131031): forward 5′-TTGCCCCGAGGCTCTCTT-3′; reverse 5′-AGTTGAAGGTGGTCTCGTGGAT-3′. The homemade Taqman probes were labeled at the 5′ with 6′ FAMTM fluorochromes for rhodopsin and VIC® fluorochrome for β-actin, while the 3′ ends were labelled with the Minor Grow Binder quencher. The qRT-PCR reactions were performed using the Taqman universal PCR Master Mix (Applied Biosystems, Foster City, CA, USA) using 5 pmol of each primer and 9 pmol of both target and β-actin probe. The assays were performed in triplicate using a 7500 PCR Real-Time system (Applied Biosystems, Foster City, CA, USA). The results were calculated through the 2−ΔΔCt algorithm against β-actin, and expressed as the n-fold difference compared to an arbitrary calibrator, chosen as a higher value than ΔΔCts.
The assays were carried out in triplicate. All experimental data are reported as mean ± S.D. Statistical analyses on body weight and BMI were performed by two-way repeated measures (RM) ANOVA; statistical analyses on fat morphometry and gene expression were performed by standard two-way ANOVA followed by Bonferroni’s correction. A P ≤ 0.05 was considered statistically significant; *, **, and ***represent P ≤ 0.05, P ≤ 0.01, and P ≤ 0.001, respectively.
Morphological and morphometric studies: analysis of zebrafish fat tissue and adipocytes The morphological studies in zebrafish were carried out on histological sections according to the procedure by Montalbano et al. [29, 33].
Results BMI and body weight measurements The examination of normalized body weight (Fig. 1a) showed a clear increase in both treated and untreated overfed groups at the end of the experimental protocol, although a greater increase was observed for OF (1.51 gr) than OF + MEL (1.33 gr). Fish fed with the equivalent of 20 mg Artemia cysts/day showed a modest decrease, with the minor value for treated fish (ctrl + MEL = 0.87 gr) with respect to untreated ones (ctrl = 0.98 gr). Interestingly, during the first 2 weeks the variation in BMI is similar for all the groups but, from that moment onward, the difference between melatonin-treated and untreated groups (ctrl vs ctrl + MEL; OF vs OF + MEL) became more evident. Normalized BMI trend (Fig. 1b) showed a similar variation, indeed major values were reported for melatonin-untreated groups (ctrl = 0.98 and OF = 1.53) with respect to melatonin-treated groups (ctrl + MEL 0.87 and OF + MEL 1.34), at the end of the 5th week. Statistical analysis was performed to evaluate significant differences among normalized weight and normalized BMI datasets. Two-way RM ANOVA was performed on ctrl vs OF, OF vs OF + MEL, ctrl vs ctrl + MEL and ctrl + MEL vs OF + MEL groups, showing significant weight differences (P ≤ 0.05) among all groups (Fig. 1a). At the end of experimental protocol, significant differences were reported for all groups: ctrl vs ctrl + MEL (P < 0.01), ctrl vs OF (P < 0.001), ctrl + MEL vs OF + MEL (P < 0.001), OF vs OF + MEL (P < 0.001). Since BMI is a composed parameter, we examined statistical significance in normalized dataset. At the end of experimental protocol, significant differences were reported for all groups: ctrl vs ctrl + MEL (P < 0.01), ctrl vs OF (P < 0.001), ctrl + MEL vs OF + MEL (P < 0.001), OF vs OF + MEL (P < 0.001). Normalized body weight (Fig. 1a) showed decreased values for ctrl + MEL (0.8739 ± 0.0425) and OF + MEL
Endocrine Fig. 1 a Normalized body weight graph showing the relative body weight vs t = 0 during the whole experimental period (5 weeks) in the four experimental groups (ctrl, ctrl + MEL, OF, OF + MEL). b Normalized BMI graph showing the relative BMI vs t = 0 during the whole experimental period (5 weeks) in the four experimental groups (ctrl, ctrl + MEL, OF, OF + MEL). Statistical analysis through twoway repeated measures (RM) – ANOVA was performed and Pvalues lower than 0.05 were considered significant. Here we highlight the statistical significance between MELtreated and matched control groups
(1.3340 ± 0.0473) with respect to ctrl (0.9801 ± 0.0582) and OF (1.5114 ± 0.0372) groups, at the end of the 5th week. Normalized BMI (Fig. 1b) showed a clear, statistically significant decrease in OF + MEL (1.3371 ± 0.0364) group with respect to OF group (1.5316 ± 0.0391) at the end of the experimental protocol (P < 0.001) as well as ctrl (0.8700 ± 0.0372) with respect to OF (1.5316 ± 0.0352). Similarly, ctrl + MEL group show an evident, statistically significant decrease in normalized BMI at the end of the 5th week (0.9801 ± 0.0260) compared to OF + MEL group (P < 0.001). A significant difference (P ≤ 0.01) between ctrl (0.8700 ± 0.0372) vs ctrl + MEL (0.9801 ± 0.0260) at the end of experiment was found. The amount of Artemia left in the fish tanks was controlled far in advance of analyzing BMI, but no differences among the experimental groups were observed (data not shown).
Morphometry of adipose tissue Morphometric analysis showed a different development of adipose tissue area in the four groups. Indeed, average area of visceral and subcutaneous adipose tissue showed a significant (P < 0.001 and P < 0.01 respectively) decrease in OF + MEL (406685.42 ± 38567.52 and 431755.57 ± 55837.05, respectively) (Fig. 2d–f), with respect to OF (907004.67 ± 89232.72 and 814093.25 ± 103195.78, respectively) (Fig. 2b, e, f); conversely, in ctrl (92973.15 ± 14456.28 and 110113.80 ± 31752.96, respectively) (Fig. 2a, e, f) and trl + MEL (72502.23 ± 13172.82 and 119810.18 ± 27896.74, respectively) (Fig. 2c, e, f) the treatment did not produce any statistically significant effect (P = 0.3345 and P = 0.8203). The average size of visceral adipocytes appeared significantly different (P < 0.001) between ctrl (1334.98 ±
Endocrine
Fig. 2 a–d Hematoxylin and Eosin staining of sagittal sections showing visceral and subcutaneous fat depots in the four experimental groups (ctrl, ctrl + MEL, OF, OF + MEL). e, f Bar graph showing the values retrieved by morphometric analysis of fat on average area of visceral (e) and subcutaneous (f) adipose tissue per section in the four
experimental groups (ctrl, ctrl + MEL, OF, OF + MEL). The values are expressed in µm2. Statistical analysis through standard two-way ANOVA, followed by Bonferroni’s correction, was performed and P values lower than 0.05 were considered significant (** and *** represent, P ≤ 0.01 and P ≤ 0.001 respectively). Scale bars (a–d) 1 mm
93.37) (Figs. 3a, i) and ctrl + MEL (626.95 ± 88.93) (Figs. 3d, i), as well as between OF (1822.89 ± 142.66) (Figs. 3b, i) and OF + MEL (1100.15 ± 61.87) (Figs. 3c, i) (P < 0.001), indicating a reduction in cell dimensions and an increase in cell number to cover the same adipose tissue area. Conversely, the average size of subcutaneous adipocytes showed a significant difference (P < 0.001) only between ctrl (1890.72 ± 294.38) (Figs. 3e, l) and OF (4450.48 ± 287.86) (Figs. 3f, l), while there were no statistically significant differences among ctrl vs ctrl + MEL (1959.83 ± 283.63) (P = 0.86761) and OF vs OF + MEL (4538.52 ± 244.87) (P = 0.81555) (Figs. 3g, h, l). The OF + MEL showed a statistically significant (P < 0.001) decrease of visceral adipocytes number (311.35 ± 28.89) compared with OF (592.50 ± 61.55) (Fig. 4a), hence demonstrating a great lipolitic action. Moreover, visceral density of adipocytes (Fig. 4c) was not significantly higher
(P = 0.12955) in OF + MEL (0.00075 ± 0.000052) than in OF (0.00064 ± 0.000041), being directly proportional to a putative lipolitic action; in other words, considering the same adipose tissue area, the number of adipocytes was higher in OF + MEL group than in OF. Statistically significant difference (P < 0.05) between ctrl (0.00053 ± 0.000091) and ctrl + MEL (0.00088 ± 0.00017) was also found, in terms of visceral density of adipocytes. The number of subcutaneous adipocytes (Fig. 4b) was approximately equal to the number of visceral adipocytes; this fact highlighted a great statistical significance (P < 0.001) between ctrl vs OF (53.30 ± 8.48 and 268.29 ± 29.21, respectively) and between OF vs OF + MEL (147.21 ± 20.16) (P < 0.001), because of the above hypothesized lipolitic action. The density of subcutaneous adipocytes (Fig. 4d), consistently to data on subcutaneous adipocytes average size, did not show statistically
Endocrine
Fig. 3 a–h Hematoxylin and Eosin staining of sagittal sections showing visceral and subcutaneous adipocyte size in the four experimental groups (ctrl, ctrl + MEL, OF, OF + MEL). i–l Bar graph showing the values retrieved by morphometric analysis of fat on visceral (i) and subcutaneous (l) adipocyte average size in the four
experimental groups (ctrl, ctrl + MEL, OF, OF + MEL). The values are expressed in µm2. Statistical analysis through standard two-way ANOVA, followed by Bonferroni’s correction, was performed and P values lower than 0.05 were considered significant (** and *** represent P ≤ 0.01 and P ≤ 0.001, respectively). Scale bars (a–h) 20 µm
significant differences in the four groups, except for ctrl vs OF (0.00059 ± 0.00011 and 0.00034 ± 0.000024, respectively) (P < 0.05).
0.001), ctrl vs OF + MEL (P < 0.001), ctrl vs ctrl + MEL (P < 0.01), OF + MEL vs ctrl + MEL (P < 0.05), OF vs ctrl + MEL (P < 0.05). In the gut (Fig. 5b), the highest leptin expression level was found in ctrl + MEL and OF groups, that showed comparable values (0.6000 ± 0.2 and 0.5100 ± 0.09504, respectively). The hormone mRNA reached the lowest level in OF + MEL (0.0400 ± 0.02) and an intermediate value in ctrl group (0.3700 ± 0.1102). Statistically significant differences in expression levels were observed among OF vs OF + MEL (P < 0.001), OF + MEL vs ctrl + MEL (P < 0.001), ctrl vs ctrl + MEL (P < 0.05), ctrl vs OF + MEL (P < 0.01).
Semi-quantitative RT-PCR (qRT-PCR) qRT-PCR was carried out on brain and gut samples to analyze the expression of orexigenic (ghrelin, orexin, NPY) and anorexigenic (leptin, POMC) genes in the four experimental groups (Figs. 5, 6). Leptin In the brain (Fig. 5a), leptin showed the highest expression level in the ctrl group (0.5400 ± 0.09504). ctrl + MEL revealed lower leptin levels (0.3200 ± 0.07506) compared to ctrl group. Leptin mRNA levels in OF and OF + MEL groups were very similar (0.1900 ± 0.05508and 0.1800 ± 0.05568, respectively). Statistically significant differences in expression levels were observed among ctrl vs OF (P
