EFFECT OF MELATONIN DIFFERENT TIME ADMINISTRATION ON THE DEVELOPMENT OF DIET-INDUCED OBESITY IN RATS

In recent years much attention has been paid for study of the melatonin use possibilities for improving obesity comorbidities. The aim of our study was to determine the influence of melatonin different time treatment on body weight changes of dietinduced obesity in rats. The administration by gavage of melatonin in dose 30 mg/kg for 7 weeks had the potential to decrease visceral fat weight, Lee index (both after morning and evening treatment) and body weight gain rate (only after evening dose).

melatonin levels are highest (during the hours of darkness), the affinity of melatonin for its receptor, total binding and the expression of melatonin receptor mRNA are lowest. In addition, when cultured or recombinant cells expressing melatonin receptors are exposed to melatonin chronically, the potency of melatonin at melatonin receptors decreases perhaps due to an uncoupling of the receptor from its effector, due to receptor internalization and/or due to receptor down-regulation [19,20,21]. However, when melatonin levels are lowest (during the hours of daylight), the affinity of melatonin for binding, total binding and the expression of melatonin receptor mRNA are highest [22].
Despite the large number of scientific papers devoted to studying melatonin for the treatment and prevention of obesity [10,11,12,13], we still lack knowleige on the use of this signal molecule, since different doses, duration, route and time of administration were taken in the researches. This dependence is due to the different sensitivity (density, affinity) of melatonin receptors throughout the day [23], but the mechanism of such regulation is still completely unclear [24].
The aim was to study body mass and food consprumision changes on the development of rat high-calorie diet-induced obesity after melatonin use and perform comparative study the treatment efficacy of melatonin evening (1 hour before light-off) vs morning administration (1 hour after light-on) for the prevention and treatment of development obesity.
Materials and methods. White nonlinear male rats weighing 100-120 g were used in this study. The light cycle was 12-h light and 12-h darkness, with lights-off at 19:00 h. All experiments on animals were carried out in compliance with the international principles of the European Convention for the Protection of Vertebrate Animals used for experimental and other scientific purposes (European Convention, Strasburg, 1986), Article 26 of the Law of Ukraine "On the Protection of Animals from Cruelty" (No. 3447-IV, February 21, 2006) as well as all norms of bioethics and biological safety.
During the first week, all animals received standard rodent chow. On the 8 th day, the animals were randomized into 2 groups: control animals received standard chow (3,81 kcal/g) for 10 weeks and experimental rats received high-calorie diet (5,35 kcal/g) consisting of standard chow (60%), pork fat (10%), eggs (10%), sugar (9%), peanut (5%), dry milk (5%) and vegetable oil (1%) [25]. Food and water were available ad libitum. To confirm the development of obesity the animals were weighed one times a week until the average body gain reached a significant difference of at least 30% between the two groups. They were then divided into 6 group according to table 1. Melatonin (Alcon Biosciences, USA) was administered daily by gavage for 7 wk (30 mg/kg) either 1 h after lights-on (Zeitgeber time (ZT) 1) or 1 h before lights-off (ZT11) (Fig. 1). Melatonin treatment was began at 6 th week of study after obesity is developed.

Fig. 1. Schematic protocol of melatonin administration
Food and water consumption were measured daily at the same time (09:00 to 10:00 h) and body weights were determined once a week. Body weight gain, relative daily food (kcal/day/g body weight) and relative daily water consumption (ml/day/g body weight) was determined for each rat. Body length was measured; body mass index (BMI) (the ratio of body weight (kg) of rats to the square of the body length (m 2 )) and Lee obesity index (the ratio of cube root of body weight (g) by nasoanal length (cm) and multiplying the result by 1000 [26]) were also calculated. The epididymal, retroperitoneal, perirenal fat pads were dissected and immediately weighed.
The statistical analysis of the results obtained was conducted using the Statistica 6.0 (StatSoft, USA) and Microsoft Excel 2010 (Microsoft, USA) software. Normality of data distribution was determined by the Shapiro-Wilks cri-terion. To assess the validity of the revealed changes, parametric (Student t-test for two-samples) and nonparametric (Mann-Whitney U-test for the independent groups) methods of variation statistics were used, the difference was significant at p<0,05. The obtained results are presented as M ± SEM (mean ± standard error of mean).
Results and discussion. To establish the obesity model, animals were fed HCD until there was a minimum difference of 30 % body weight gain between rats fed HCD compared with those who were fed standard diet. As soon as this difference reached significance (p < 0.05), parts of HCD and standard diet fed rats were treated with melatonin either 1 h after lights-on (ZT1) or 1 h before lights-off (ZT11). The baseline information of body weight, related visceral fat weifht, body mass index, and Lee index of experimental animals are presented on the table 2. The weight gain, BMI and Lee index in rats received standard chow, which administreted melatonin (M ZT01 and M ZT11), were not significantly different compared with control group, but the level of visceral fat weight was decrease (p < 0.05) in M ZT01 by 48% and in M ZT11 by 47%. However, the data in literature about melatonin influence on body weight in rats with normal type of diet are disputed: was shown decrease (continuously in drinking water for 12 and 24 weeks in dose 0.4 μg/mL [27], or for 9 weeks in dose 25 μg/mL [28], or for 3 and 6 weeks in dose 4 mg/kg/day [29]) or no effect (intraperitoneal injection for 6 months at 09:00 hr in dose 3 mg/kg/day [30], or for 2 weeks at 07:00 hr in dose 1 and 10 mg/kg/day [31], or for 5 weeks at 18:00 hr in dose 10 mg/kg/day [32], or continuously in drinking water for 10 weeks in dose 25 μg/mL [33], or for 8 weeks in dose 100 mg/kg/day [34]). In our study reduce in visceral fat weight in the absence of significant differences in food intake (data not shown) are worth exploring. A key piece of evidence in this regard is the observation that melatonin plays a role in seasonal changes in adiposity by increasing the activity of the sympathetic nervous system innervating white fat which leads to lipolysis [35].

T a b l e 2. Body weight gain, body mass index, Lee index and visceral fat weight of experimental animals
The weight gain and BMI in HCD ZT01 and HCD ZT11 groups take intermediate value: there no significant difference compare both to control and HCD group. If we pay attention to dynamics of weight gain (Fig. 2), all this data means that melatonin have influence to body mass changes and tendency to decrease weight gain during development of obesity. After 1 weeks of melatonin administration (on the 7 th and 8 th weeks of experiment) the weight gain of HCD ZT01 and HCD ZT11 groups begin stop growing and this tendency was maintained during following final 5 weeks (decreases in HCD ZT01 by 6% and in HCD ZT11 by 8% groups in relation to HCD, but still increased by 30% and 27% in HCD ZT01 and HCD ZT11 respectively in relation to control, to note HCD weight gain was higher by 40% than control). Surprisingly, we observed significant difference in weight gain rate (Fig. 3 A, B). At 2 nd and 3 th weeks after melatonin treatment (8 th and 9 th weeks of experiment) the weight gain rate were reduced both in HCD ZT01 by 50% and 29%, in HCD ZT11 by 38% and 67% (р < 0.05 compared with HCD). Lower values in HCD ZT01 at 1 st weeks melatonin use (by 73% р < 0.05 compared with HCD) we explain the stress action under first week of morning administration by gavage, because the same raise we mark in M ZT01 by 62% (р < 0.05 compared with control). Anyway, except 1 st week melatonin effect on weight gain rate in M ZT01 and M ZT11 groups was without significant difference throughout the experiment in compared with control (data not shown).  The following 4 th and 6 th weeks (10 th and 12 th weeks of all experiment) we found reduce value of this parameter in HCD, HCD ZT01 and HCD ZT11 groups to control level due to low quality of hight-fat chow (this points is outpoints).
The next 5 th and 7 th weeks (11 th and 13 th weeks of all experiment) the weight gain rate in HCD ZT11 continue loss to control values (by 72% and 57%, р < 0.05 compared with HCD), while in HCD ZT01 studied parameter did not significantly differ from HCD group.  Weight gain rate, % weight gain rate was in HCD elevated by 60% in compare with control (р < 0.05)). These data may be suggest about more efficient influence on the body weight gain rate by melatonin evening dose. The information about Lee index and visceral fat weight also provide that melatonin amelioreted body mass dynamic under obesity development (table 2). According to obtained results we have revealed an increase Lee index of HCD group by 7% (p < 0.05, 309 vs 288) compared with control rats. Study shown that Lee index in HCD ZT01 and HCD ZT11 decrease by 4% and by 4,5% respectively (p < 0.05) compared with HCD (297, 295 vs 309), and were similary to control level after 7 weeks of melatonin treatment. The values of relative visceral fat weight also indicate efficient action of melatonin application on body mass under hight-calorie diet condition. The index of studied parameter in HCD attained values by 64% higher than controls. After melatonin use in HCD ZT01 and HCD ZT11 the relative visceral fat weight have fall to control level, at the same time it was decrease by 38% and 46% as compared with HCD group.
Interestingly, the changes associated with body mass (weight gain, weight gain rate, BMI, Lee index, visceral fat) happend independly of food and water consumption (Fig. 5, 6). As shown the control group consumed an average of 0.247±0.002 kcal/g of standard chow per day. The HCD rats ate an average of 0.337 ± 0.006 kcal/g of highcalorie food, which higher (strongly marked hyperphagia)by 37% (p< 0.05) than control rats. However, the rats in HCD ZT01 and HCD ZT11 ate an average of 0.327 ± 0.005 kcal/g and 0.347 ± 0.006 kcal/g of feed accordingly, which actually does not differ from the values of the HCD group, but elevated by 32% and 40% than control (p< 0.05). The relative daily food consumption in M ZT01 and M ZT11 were similar to control values and did not different statistically (0.258 ± 0.005 and 0.245 ± 0.003).

Fig. 6. Effect of melatonin treatment on relative daily water consumption
Studies have found that HCD rats consumed an average of 32.2 ± 0.4 ml/g of water per day, which is lower by 17% (p < 0.05), whereas control group drink 38.9 ± ± 0.8 ml/g. The relative daily water consumption in HCD ZT01 and HCD ZT11 were lower (p< 0.05) by 22% (30 ± ± 0.6 ml/g) and by 14% (33.4 ± 0.7 ml/g) than the control group and did not differ from HCD group values. Melatonin administration significantly did not affect water consumption in M ZT01 (39.3 ± 0.9 ml/g) and M ZT11 (39.3 ± 0.6 ml/g).
This data in agreement with other researches: after melatonin treatment (continuously in drinking water for 8 weeks in dose 100 mg/kg/day) was demonstrated significance decrease visceral fat weight in obese (ob/ob) mice, while weight gain have intermediate position compare to control and obese animals [36]. But, on the other hand, under condition of HCD the concomitant administration (continuously in drinking water) of melatonin significantly attenuated a body weight increase without affecting chow or water consumption [33,11,37]. These data indicate that melatonin have other mechanisms to influence on body weight -stimulates the appearance of beige adipocytes (mixed type) in white adipose tissue [38] (which are contribute to the loss of excess accumulated triacylglycerides due to heat production); normalizes circadian secretion of adipokins [39]; shows anti-inflammatory [40] and antioxidant properties [41]; involvement in the regulation of appetite and intake of food through the influence on the hypothalamus nucleus [42]. Except intermediate and lower level of weight gain after melatonin use, there are information about "no effect" -in this studies was use reduce dose or period of administration [31,29] (but also was shown visceral fat weight decreased), or another route of administration [43,44].
A limitation of this study may be that no significative difference in HCD body weight gain was seen after 7 weeks of melatonin treatment; a longer duration of treatment may be needed to elicit such improvement or increased rats numbers in groups. Moreover, probably muscle weight gain would be interesting, since it have been shown raise in muscle Ferets diameters after melatonin use, which may also make contribution in body weight [45].
In the light of chronobiological question time of administration, our data suggest that evening melatonin administration improve visceral fat and body weight parameters more rapidly then morning. We hypothesized that morning dose may prolong night melatonin secretion peak to support its highest level for longer time, while evening dose was intended to raise endogenous night peak. However, weight gain, BMI, Lee index and visceral fat weight are lower and have more stronger expressed difference in HCD ZT11 then in HCD ZT01. Previous study shown the same results: melatonin effects on body weight in a model of overweight were possibly time dependent -the first melatonin administration (ZT 04) was less efficiant then in ZT 11 (also reduces weight gain) [46]. Subcutaneous (or intraperitoneal) injection 2-3 h before lights-off decreased body weight and visceral fat too under influence middle dose of melatonin (1 or 4 mg/kg/day for 4 or 8 weeks) during developmental obesity [47,48] and besides intra-peritoneal injection in the morning at the same dose have no effect to body weight [31].
Conclusions. The application of melatonin by systemic administration led to reduction of developed obesity complication associated with body weight. If we take into consideration all studied parameters (body weight gain, weight gain rate, BMI, Lee index and related visceral fat weight), we make decision about more profitable use melatonin administration 1 h before lights-off then 1 h after lights-on. Moreover, the changes of these parameters did not connect with lower food consumption. This changes may be provide by stabilizing balance of anti-and proinflammation reaction, normalising circadian rhythm of adipokins secretion, appearing beige adipocytes, stimulating brown adipocyte differentiation, etc.