THE EFFECTS OF MELATONIN ADMINISTRATION IN DIFFERENT TIMES OF DAY ON THE BROWN ADIPOSE TISSUE IN RATS WITH HIGH-CALORIE DIET-INDUCED OBESITY

The aim of our study was to determine morpho-functional state (area of nucleus, brown adipocytes and also area and number of lipid droplets in each cells, general optical density of tissue) of brown adipose tissue in rats with high-calorie (high fat) diet-induced obesity after melatonin administration in different time of the day (morning and evening). Melatonin was administered daily by gavage for 7 weeks in dose 30 mg/kg either 1 h after lights-on (ZT01) or 1 h before lights-off (ZT11) rats with high-calorie diet (HCD). Besides morphometric parameters as well were measured related visceral fat weight and related brown adipose tissue mass. Rats with HCD had huge changes in brown adipocytes morphology, which summarized in become resembles of classical white adipocytes: grown lipid droplets and cells area, but goes down lipid droplets number and optical density of brown adipose tissue. In general brown adipose tissue with above mentioned characteristic from HCD rats lose their ability to conduct strongly thermoproduction function. After melatonin used in rats with HCD arise leveling of pathological changes, which associated with consumption of HCD. Namely, in groups HCD ZT01 and HCD ZT11 we obtain decreased cells and lipid droplets area, increased lipid droplets number and optical density of brown adipose tissue, in relation to group HCD. Therese received changes has evidence about functionally active brown adipose tissue state, which can also dissipate of exceed energy (lipids – triacylglycerols) amount into whole organism during heat production for avoid to its storage in white adipose tissue and in outside adipose tissue. In addition, evening administration of melatonin (group HCD ZT11) demonstrate more activated state of brown adipose tissueand also related visceral weight gain less, than morning(group HCD ZT01). In conclusions, melatonin influence on morpho-functional state brown adipose tissue in rats with HCD, moreover evening administration can use for obesity therapy via its strong action on activate brown adipocytes.

Introduction. The overweight and obesity become wide medical problem, since were shown its involvement in development of 2 type diabetes [1]. The number of people with overweight and obese are grown every year [2]. Potentially, this people fall into risk diabetes group [3]. The search continues for effective and safe methods to prevent obesity. One of way to treatment obesity is target activated brown adipose tissue (BAT) [4,5]. BAT through decrease fat mass, increase glucose uptake ameliorate insulin sensitivity and prevent 2 type diabetes development [6,7].
Brown adipose tissue (BAT) -connecting tissue with specific function of lipids deposition for the heat production [8]. Since 2007 when activated BAT were open in adult human, its presence induce a huge interest [9,10]. Usually, BAT activated after cold exposure or sympathetic influence of adrenaline [11,12]. But these methods of initiate BAT have many side effect (for example, adrenaline on cardiovascular systems). So, actual is finder agents to trigger BAT without (or with minimize) side effect for obesity improve [13,14]. One of the potentiate candidate can be melatonin.
In the other hand was demonstrate presence of melatonin receptor on brown adipocytes [23,24]. Melatonin provoke in brown adipocytes expression of uncoupling proteins UCP1 in mitochondria [25] and induction thermogenesis [26,27]. Thus effect may be beneficial for obesity therapy [28]. Now grow intensify studying about chronotherapy uses for cure most of illness, including obesity [29,30]. It is need more dedicated study of chronopharmacological agents, including melatonin, in the treatment of metabolic diseases [31].
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 %), lard (10 %), eggs (10 %), sugar (9 %), peanut (5 %), dry milk (5 %) and vegetable oil (1 %) [32]. 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 and the respective animals were classified as having the normal body mass (Control) and those with development of obesity (HCD). Rats of HCD groups were divided into tree subgroups: one subgroup received no MT, animals of the second and third subgroups obtained different modes of MT administrations -single peroral by gavage introductions, 1 h after light switching on (group HCD ZT01) and 1 h before light switching off (group HCD ZT11) (modes "morning" and "evening", respectively). Thus, the experimental subgroups are indicated below as normal body mass (control), HCD, HCD ZT01, HCD ZT11.
Melatonin (Alcon Biosciences, USA) was administered daily by gavage for 7 wk (30 mg/kg) either 1 h after lightson (Zeitgeber time (ZT) 01) or 1 h before lights-off (ZT11). Melatonin treatment was began at 6th week of study after obesity is developed.
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.
On the last day of the experiment, the animals were decapitated, and then the brown adipose tissue was isolated from the interscapular region and weighed for the calculation of the relative mass. Also the visceral (epididymal, retroperitoneal, mesenteric, perirenal) fat pads were dissected and immediately weighed.
Histological examination was performed to characterize the morphology and functional status of brown adipose tissue. Fragments of brown adipose tissue in the size of 1x1 cm were fixed in 4 % of paraformaldehyde in 0.1 M phosphate buffer for 72 h, after which they were dehydrated and embedded into paraffin according to a standard procedure. From the paraffin blocks, 5 μm sections were performed and stained with Bemer's hematoxylin and eosin. Further examination of sections was performed using a light microscope BX41 (Olympus, Japan). Microphotographs were taken using the DP20 (Olympus, Japan) digital camera and the QuickPHOTO MICRO software (Promicra, Czech Republic).
The cross-sectional area of the nucleus, cells and lipid droplets, nuclear-cytoplasmic ratio (NCR), the number of lipid inclusions per one cell and the optical density of the tissue were used as criteria for assessing the morphology and functional status of brown adipocytes. All parameters were measured using the ImageJ software (National Institutes of Heath, USA).
Statistical data analysis was performed using the Statistica 6.0 (Stat-Soft, USA) and Microsoft Excel 2010 software (Microsoft, USA). The distribution of values was estimated using Shapiro-Wilk W-test. Since the deviation of these values distribution of from the normality was minor, to evaluate the differences between the values we used Student's t-test. The differences with probability of the null hypothesis p < 0.05 were considered significant. The obtained results are presented as the mean ± standard error of mean.
Results and discussion. In control group (Fig. 1 A) brown adipocytes have polygonal cell morphology with round-shaped central-located nucleus. In cytoplasm accommodate a few amount droplets of lipid (Fig. 1,  asterisk), which did not stain with water-soluble dyes, thats why they look like white bubble. Another part of cytoplasm, which contain a large number of mitochondria, show eosinophilic properties. After consumption HCD (Fig. 1 B) brown adipocytes become resembles morphology of white adipocytes: round cells with peripheral-located oval nucleus, because practically unilocular lipid droplet occupy almost all space of cytoplasm. Described changes indicate about decrease of brown adipocytes functional activity [33,34]. If melatonin used at morning time of day (group HCD ZT01, Fig. 1 C), adipocytes represent improvement, although the shape of cells remain circularized, nucleus become at the centre of cell and accordingly lipid droplets size goes down, also their number rise. But, melatonin administration at the evening time of day (group HCD ZT01, Fig. 1 D) give a more stronger effect: adipocytes in line with control have polygonal cell morphology and round central nucleus (characteristics, which completely reverse manifestation of obesity-associated phenotype of brown adipocyte), they demonstrate maintain multilocular little lipid droplets. Thats changes have marked pronounced functional activation of brown adipocytes thermogenesis [35]. In HCD group the area of brown adipocytes (Fig. 2) was increase by 30 %, but in case melatonin administration this parameter decrease in group HCD ZT01 by 16 % and in group HCD ZT11 by 50 % (compare to control). In turn in rely to HCD group melatonin provoke significant reduction in group HCD ZT01 by 35 % and in group HCD ZT11 by 60 %. Besides, was difference between HCD ZT01 and HCD ZT11 groups by 40 %. It can be explain by changing their function during obesity development -to deposit exceed lipids, that why they have rise in size in HCD group and become as white adipocytes [36]. In continuation of this pathologic changes under diet-induced obesity (group HCD) adipocytes undergo to magnification area of lipid droplets by 44 %, and melatonin application decline its by 28 % in HCD ZT01 and by 61 % in HCD ZT11 both in relation to control level; that is in compare to HCD group by 50 % and 73 % in HCD ZT01 and HCD ZT11, respectively, (also have fixed significant difference between morning and evening administration by 47 %). Obtained data demonstrate starting lipolysis process after melatonin application, moreover most effectively it manifested in HCD ZT11 group, which can be connected with targeting REV-ERB nuclear receptor by melatonin [37] (which involve in regulate circadian rhythm expression of a key lipolysis enzymes in adipocytes [38]), through its more sensing because REV-ERB mRNA high level in brown adipocytes is fixed at the beginning of night [39].
In the other hand, the number of lipid droplets did not change during obesity development. Intriguing, that in group HCD ZT11 (evening melatonin administration) lipid droplets amount rise in relation to control by 16 % and in compare with HCD by 22 %. Similar results were obtained after melatonin uses in rats with standard diet [40]. This effect connected with activation of melatonin receptor in brown adipocytes and enhance lipolysis, as well as UCP1 producing [41,42]. Also was shown that increase lipid droplets number and decrease their area are testify about strong activity of cells in heat production [43].
At standard histological tissue processing, lipids, which are deposited in the adipocytes in the form of inclusions, are washed out during the dehydration of the tissue. Therefore, the places of their localization in the cytoplasm of the cell after staining with eosin remain uncoloured. At a smaller magnification of the lens, it is possible to estimate the overall brightness, which depends directly on the amount of lipids in the tissue -optical density of brown adipose tissue. As HCD group consume high-calorie diet and consequently has enriched of lipid adipocytes, tissue shown less optical density by 34 % in relation on control. After melatonin induction the optical density reverse to level of control and, respectively, grow by 45 % in HCD ZT01 and by 46 % in HCD ZT11 without no significant difference between group with morning/evening administration melatonin. Thats data suggest about decrease lipid content in brown adipocytes and also increased eosinophilic part of cytoplasm. Its can be in relation with mitochondria amount grown, because melatonin influence on mitochondria metabolism due to localization on mitochondria membrane melatonin receptors MT1 and MT2 [44,45]. In general, melatonin administration influence on fat deposition and body weight (Table 1) [46,47]. There no significant difference change in related brown adipose tissue mass, which can be explain functional rebuilding of tissue: in HCD lipid droplets content amplify, while in HCD ZT01 and HCD ZT11 enlarged respectively cell's number, which connected with enhance functional activity adipocytes.The literature data is contradictory about change brown adipose tissue mass after melatonin intervention: some shown itsincreased activity and volume grown [48,49], other -decreased mass, but rise of activity too [50]. Presented different data can be depend on various dose, route and time of melatonin administration.  During consumption of HCD the related visceral adipose tissue weight significant grow in compare with control by 65 %, that is confirm developed obesity. Morning melatonin introduction lower related visceral adipose tissue weight by 28 % in compare with HCD group, and by 18,5 % in compare with control. Thus, in HCD ZT01 group related visceral adipose tissue mass has intermediate place: its significant differ both from control and HCD group. Evening melatonin administration reduce related visceral adipose tissue weight by 39 % in rely to HCD and reach control value. Shown changes occur without any alterations in food and water consumption.
Conclusions. High-calorie diet change morphofunctional state brown adipocytes, closer their phenotype to white adipocytes. Melatonin treatment improve brown adipose tissue state (adipocyte size and lipid contain) in rats with diet-induced obesity, moreover after evening administration brown adipocyte activity increased in obese rats even in comparison with non-obese. Also activated brown adipose tissue may be one of the way to spread excess lipids, since related visceral adipose tissue mass decrease without reduction food and water consumption. 2. Global, regional, and national prevalence of overweight and obesity in children and adults during 1980-2013: a systematic analysis for