Inflammation features of brown adipose tissue of rats with diet-induced obesity development after different regimes of melatonin administration

One of the prominent obesity-related changes is the development of systemic low-grade proinflammatory state. Brown adipose tissue (BAT) may serve as a potential target for activation by melatonin to facilitate heat production and simultaneouslystimulate lipolysis during obesity development.At the same time, melatonin is known to have immunomodulatory properties, which are performed via endocrine and paracrine signal pathways in variety cell types (including brown adipocytes)and change significantly during the day. Therefore, it can be used in a wide range of doses and at different times of the day (chronotherapeutic approach). Thus, the main goal of our research was to analyze the inflammation state of brown adipose tissue of rats during high-calorie diet induced-obesity development after different daily melatonin application in different regimes. Melatonin was administered by gavage for 7 weeks in dose 30 mg/kg 1 h before lights-off (HCD ZT11, M ZT11, evening), or 1 h after lights-on (HCD ZT01, M ZT01, morning). Tissue collagen contain and leukocyte infiltration levels in BAT, detected by Van Gieson trichrome staining, were used as markers for assessment of BAT inflammation state BAT.Propagation of obesity resulted in the increase of BATfibrosis level (the relative area occupied by collagen fibers) and tissue leukocyte infiltration in comparison to control rats. BAT fibrosis level after melatonin administrations to obese rats of HCD ZT01 and HCD ZT11 groups decreased to control values.Similar effectswere observedinBAT tissue leukocyte infiltrationafter both regimes (HCD ZT01 and HCD ZT11 groups) of melatonin intake: this parameter decreased significantly, comparing to obese rats, but was still elevated, comparing to controls. At the same time, melatonin treatmentin morning or evening regimes did not have any impact on BAT fibrosis propagation and leukocyte infiltration in animals that consumed standard diet (M ZT01 and M ZT11 groups). To sum up, we suggestcorrectivepropertiesof melatonin in context of chronic low-grade inflammation in obese rats BAT and suppose its wide potential for the therapeutic usecombined withvirtually absent side effects on BAThistophysiology of non-obese rats.


Introduction.
Obesity is associated with an increase in size (hypertrophy) and de novo formation of white adipocytes (hyperplasia) [1]. Obesity-related changes occur not only in white adipose tissue, but also in brown adipose tissue (BAT). These changes are manifested in several processes, which are collectively named "whitening" of BAT [2]. During "whitening" brown adipocytes dramatically change their morphology to a white adipocytes-resembling phenotype [3]. The increase in adipocytes' volume and tissue mass causes inflammation, further deregulation of adipose tissue and, ultimately, adipose tissue fibrosis [4], which is manifested in obesity and diabetes. The main inflammatory cytokines, whose mRNAs are detected in BAT of obese animals, are TNFα [5], F4/80 antigen -a major murine macrophage marker [6], monocyte chemoattractant protein 1 [7]. High levels of inflammatory molecules can lead to inhibition of insulininduced glucose uptake and to the development of insulin resistance in BAT [8]. Alternatively, pro-inflammatory cytokines decrease UCP 1 (uncoupling protein 1) expression in brown adipocytes, which results in decline of heat production capacity through different signal transduction pathways, possibly including SIRT1 (Sirtuin 1 -NAD-dependent protein deacetylase) and its negative regulator DBC1 (deleted in breast cancer 1) [9]. In addition, TNFα may serve as a potential candidate for thermogenesis inhibition in brown adipocytes [10]. Another, indirect, mechanism was proposed -pro-inflammatory cytokines recruit inflammation-driven macrophages to BAT, which affects norepinephrine sensitivity of brown adipocytes (the main activation mechanism of UCP 1 production) by inflammasome-mediated pathway. These changes may involve regulation of monoamine oxidase A (a catecholamine degrading enzyme) and norepinephrine transporter, that influence on norepinephrine bioavailability [11,12]. The long-term consumption of high-fat diet also results in attraction of lymphocytes and granulocytes to BAT (besides macrophages and monocytes), which promotes pro-inflammatory state in tissue [13,14].
Like other fibrotic diseases, adipose tissue fibrosis is the accumulation and increased production of extracellular matrix proteins [15]. Several factors, like leukocytosis, enlargement of adipocytes, and overproduction of HIF (Hypoxia-inducible factor) promote deposition of collagen fibers via upregulation of pro-fibrotic genes such as lysyl oxidase, collagens I and III [16,17]. Fibrosis also decreases flexibility of extracellular matrix and remodeling abilities of adipose tissue, which are associated with comorbidity of metabolic diseases [18].
Melatonin is a multifunctional signal molecule with pronounced immunomodulatory function [19]. In particular, melatonin possesses anti-inflammatory properties in obesity treatment [20]. Melatonin receptors are found in brown adipocytes [21] and in many leukocytes [22]. Main effects of melatonin on BAT are caused by modulation of UCP 1 production and mitochondrial functionality [23]. However, effects of melatonin usage on brown adipose tissue remodeling during obesity and corresponding fibrosis development remain largely unknown.
Therefore, the main aim of our research was to analyze the inflammation state of brown adipose tissue of rats (fibrosis level and tissue leukocytes infiltration) after different melatonin regiments against the background of high-calorie-diet-induced obesity.
Materials and methods. White nonlinear male rats (110±10 g bodyweight) were used in this study. The light cycle was set as 12-h light and 12-h darkness, with lightsoff at 19:00 (ZT12). 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.
Melatonin (Alcon Biosciences, USA) was diluted in drinking water and administered daily by single oral 2 mL gavage in the dose of 30 mg/kg bodyweight. The administrations lasted for 7 weeks. Melatonin treatment began at the 6th week after the propagation of obesity.
Different doses, methods and times of melatonin administration werepreviously shown to be effective in many experimental diseases models [25] and alsoclinical trials [26]. We chose the lowest dose of melatonin whichcauses both a decrease in obese rats' weight gain and the appearance of beige adipocytes, as we were interested in obesity therapy through beige and brown adipocytes' activation.
On the last day of the experiment, animalswere sacrificed by carbon dioxide asphyxiation and decapitated, and then brown adipose tissue samples were isolated from the interscapular region.
Histopathological examination was performed to characterize the morphology and inflammation status of BAT. Fragments of BAT in the size of 5 × 5 mm were fixed in 4 % paraformaldehyde in 0,1 M phosphate buffer for 72 h, dehydrated, embedded into paraffin and cut into 7 µm sections according to standard procedures.
Tissue fibrosis (estimated as relative area occupied by collagen fibers) and tissue leukocyte infiltration were used as criteria for the assessment of inflammation status of BAT. Collagen fibers were stained by Van Gieson trichrome with hematoxylin as a counterstain [27]. Quantitative determination of tissue collagen content was performed on digital microphotographs. All captures were obtained using a light microscope BX41 (Olympus, Japan) with 4×objective lens. Microphotographs were takenwith DP20 digital camera (Olympus, Japan) and QuickPHOTO MICRO software (Promicra, Czech Republic).The field of view of each subsequent microphotograph was moved to the right, retaining 20 % of the previous field until the entire specimen was taken. To create one large composite panoramic image, the cross-linking software Adobe Photoshop CS6 (Adobe Systems, USA) was used. Area of the red-stained region, which corresponds to collagen fibers was calculated and expressed as a percentage of the total area [28]. Histological evaluation of the inflammation level in the BAT was performed according to the generally accepted semi-quantitative score scale of leukocyte infiltration: absence -0, insignificant -1, medium -2, noticeable -3 [29]. We investigated at least 5 similar areas on each section. All parameters were measured with ImageJ software (National Institutes of Health, USA).
Statistical data analysis was performed using the Statistica 6.0 (StatSoft, USA) and Microsoft Excel 2010 software (Microsoft, USA). The obtained data was presented as mean ± standard error of mean (SEM).The distribution of data was assessed with Shapiro-Wilk normality W-test. Since the analyzed distributions were considered normal, we used one way ANOVA followed by Dunnet`s multiple range post-hoc test to evaluate the differences between means. The differences with probability of the null hypothesis p < 0,05 were considered significant.
Results and discussion. Brown adipocytes in control group showed typical morphology: polygonal shape, rounded nuclei that occupy a central position in cell and many tiny-sized lipid inclusions in cytoplasm (Fig. 1). Large collagen deposits were localized in parts of capsule and trabeculae of BAT. Collagen fibers were mostly present around blood vessels. We did not find any significant collagens accumulations between brown adipocytes. Similarly, substantial collagen deposits were absent near white adipocytes, which surround BAT and continuously underlie its peripheral part. Development of obesity was accompanied by the change of brown adipocytes' morphology: round shape of the cells, flat nucleus in eccentric position and unilocular massive lipid droplet, which corresponds to a white adipose tissue appearance [30]. Collagen deposits in HCD group expanded markedly in interlobular zone and near blood vessels (Fig. 1, red arrow). We detected collagen as zones of red staining, while yellow coloring indicated little to no collagen content. Intriguing, BAT collagen fibers of obese rats were closely located to some brown adipocytes, to the majority of "whitening" brown adipocytes and to many peripherally located white adipocytes. In addition to enlarged collagen fibers, in HCD group we detected presence of leukocytes (macrophages and lymphocytes) in BAT (Fig. 1, arrowheads).
Melatonin administration (M ZT01 and M ZT11 group) did not affect BAT collagen content in both trabecular and intercellular spaces of rats which received standard diet (Fig. 2). Also, we did not detect visible tissue leukocyte infiltration. We have previouslydescribed the most noticeable effects of melatonin application on morphofunctional state of brown adipocytes in rats, consumed standard diet [31]. It was mainly manifested in increased number of lipid droplets per adipocyte, cross-section area of the nucleus of brown adipocytes (and, respectively, nuclear-cytoplasmic ratio of brown adipocytes was also elevated), optical density of BAT, whereas crosssectional area of lipid droplets in brown adipocytes decreasedsignificantly.  Distribution of collagen fibers in groups HCD ZT01 and HCD ZT11 (Fig. 3) was different from control group (red arrows) -it was elevated in interlobular zone and near blood vessels, but still remarkably smaller than in HCD.
However, we did not observe massive collagen fibers that surround each individual adipocyte as in central and in peripheral zones of BAT. In addition, rare clusters spreading of leukocytes infiltration in BAT were detected.  Morphometric analysis of obtained data demonstrated significant 2-fold increase in BAT fibrosis level of obese rats after consuming high-calorie diet comparing to control (Fig. 4). Melatonin administration during HCD-induced obesity prevented the increase of BAT collagen content after both morning and evening interventions: fibrosis level in HCD ZT01 group decreased by 56 % and by 44 % in HCD ZT11 group in comparison to HCD group. The fibrosis levels in HCD ZT01 and HCD ZT11 groups did not differ from control value. There was no significant difference between morning and evening regimes of melatonin administration in HCD ZT11 and HCD ZT01 groups in tissue collagen content.Alsovariations in melatonin regimes did not influence on BAT fibrosis development in rats consuming standard diet.
HCD-induced development of obesity resulted in significant 5-fold tissue leukocyte infiltration, comparing to control group (Fig. 4). As obesity is usually accompanied by low-grade chronic pro-inflammation state, the increased tissue leukocyte infiltration was also detected in HCD ZT01 and HCD ZT11 groups after melatonin administration (3,4-fold in HCD ZT01 and 2,2-fold in HCD ZT11 groups). However, this parameter was significantly lower than in HCD group (HCD ZT01 -1,5 times, HCD ZT11 -2,3 times). Additionally, we detected significant difference between morning and evening regimes of melatonin administration in tissue leukocyte infiltration levels: it was lower by 55% in HCD ZT11, compairing to HCD ZT01.Melatonin treatment in morning or evening regimes did not influence on BAT tissue leukocyte infiltration in groups consuming standard diet.
Taking into account the results of morphological observations and morphometric analysis, melatonin was shown to have the corrective effect of on the BAT inflammation state during obesity development without any destructive influence on BAT of rats, consuming standard diet.

Fig. 4. Brown adipose tissue fibrosis level and tissue leukocyte infiltration
Notes. * -difference between the control and experimental groups are significant at p ≤ 0,05; # -difference between the HCD group and HCD ZT01, HCD ZT11 is significant at p ≤ 0,05; & -difference between the HCD ZT01 and HCD ZT11 groups is significant at p ≤ 0,05.
Our data generally correspond to previously demonstrated increase in brown adipose tissue mass and functional activity in Zücker diabetic fatty rats. These animals were treated for 6 weeks with melatonin in drinking water in the dose of 10 mg/kg body weight. The consumption of melatonin took place mainly at night as rats are nocturnal animals [32]. Unfortunately, there is no similar study with investigation of melatonin effects on BAT fibrosis under HCD-induced obesity. Itmay be related to a work that showed lower BAT fibrosis levels comparing to white adipose tissue, which was demonstrated on high-fat dietinduced obesity in C57BL6/J mice model [33]. A huge research limitation associated with the use of C57BL6/J mice is that this strain is notoriously known for being deficient in the biosynthesis of melatonin [34] and short-term lasting obesity model. Another study on diet-induced obesity showed significant alterations of BAT molecular networks associated with immune cell trafficking, lipid metabolism and connective tissue development after 24 weeks of diet consumption [35]. High fat diet-induced obesity resulted in reduction of UCP1 levels and overexpression of proinflammatory genes; while aerobic exercise led to the growth of BAT and the upregulation of anti-inflammatory genes in obese mice via cyclooxygenase 2 in the VEGF pathway [36]. Another model of diet-induced obesity in rats established development of BAT fibrosis and modulation of this parameter by intermittent food restriction [37].