Effects of physical activity and melatonin on brain‐derived neurotrophic factor and cytokine expression in the cerebellum of high‐fat diet‐fed rats

Abstract Aims Obesity suppresses brain‐derived neurotrophic factor (BDNF) expression and increases the expression of pro‐inflammatory cytokines. Herein, we assessed whether exercise training (ET), melatonin administration (MT), or their combination can affect the expressions of BDNF and cytokines in the cerebellum of high‐fat diet (HFD)‐fed rats. Methods Wistar rats (4 weeks old) were divided into five groups: normal diet (ND)‐fed control (ND‐SED), HFD‐fed control (HFD‐SED), HFD‐fed ET (HFD‐ET), HFD‐fed MT (HFD‐MT), and HFD‐fed MT plus ET (HFD‐ETMT) group. The rats were fed ND or HFD for 17 weeks. Rats were subjected to ET (running on a treadmill) and/or MT (melatonin 5 mg/kg body weight, i.p.) for 9 weeks, 8 weeks after beginning the diet intervention. Changes in BDNF and cytokine expression levels were determined using immunoblotting and cytokine arrays, respectively, 36 hours following the last bout of ET. Results Neither HFD‐ET nor HFD‐MT rats exhibited enhanced BDNF expression in the cerebellum, but HFD‐ETMT rats had higher level of BDNF expression compared with the others. The expression of TrkB, a BDNF receptor, was higher in HFD‐ETMT rats than in HFD‐ET and HFD‐MT rats. HFD enhanced the expression of interleukin (IL)‐1, IL‐2, and interferon‐γ but reduced the expression of IL‐4, IL‐6, and IL13. ET and ET plus MT counteracted these HFD‐induced changes in cytokine expressions. Conclusion Exercise in combination with melatonin confers the potential benefits of increasing BDNF and improving HFD‐induced dysregulations of cytokines in the cerebellum.


| INTRODUC TI ON
Recent studies suggest that obesity and high-fat diet (HFD) feeding with peripheral inflammation lead to deterioration in cognitive function and neurogenesis, probably via both, the dysregulation of brain-derived neurotrophic factor (BDNF) and the increase in brain inflammation. [1][2][3] Therefore, it is important to verify the effects of behavior and pharmacological interventions for improving obesity on both, brain BDNF and inflammation levels.
Interventions such as exercise training (ET) and melatonin administration (MT) have been shown to increase BDNF levels in the mouse hippocampus, [4][5][6] and MT reportedly potentiates ETinduced neurogenesis in the rodent hippocampus. 7 It can, therefore, be postulated that the combinatorial effect of ET and MT to enhance brain BDNF levels is greater than that of their individual effects. However, a verification is warranted on whether a combination of ET and MT can enhance BDMF level in brain region(s) besides the hippocampus in obesity. Moreover, there is little evidence on whether ET or MT, or both combined, can ameliorate HFD-induced brain inflammation.
The cerebellum is one of regions directly engaged in locomotor control, and it has recently been shown that a 3-week running exercise regimen brought the experimental groups' depression-associated low cerebellar BDNF levels on par with the control group's. 8 Furthermore, improvement of cerebellar inflammation has been shown to revert inflammation-induced depression-like behaviors. 9 Based on the above, we selected the cerebellum and hypothesized that ET combined with MT may be an efficacious intervention against obesity-related changes in cerebellar BDNF and inflammation levels. To this end, we assessed whether ET, MT, or a combination of both, could affect the expression of BDNF, its tyrosine kinase receptor B (TrkB), and cytokines in the cerebellum of HFD-induced obese rats.

| Animals and intervention program
Male Wistar rats (4 weeks old: SLC) were housed in a room at 23°C with a 12:12-hour light-dark cycle. All animals were divided into five groups (four rats in each group): normal diet (ND)-fed sedentary Exercise training and MT were started 8 weeks after the beginning of dietary intervention. HFD-ET and HFD-ETMT rats ran on a treadmill (5-degree incline), 5 d/wk, for 9 weeks according to a protocol reported. 10 The running time and speed were increased progressively until after 6 weeks, when the rats ran continuously for 90 minutes at 30 m/min. HFD-MT and HFD-ETMT rats received an intraperitoneal injection of MT at 5 mg/kg body weight for 9 weeks. The dose of melatonin administered was based on previous studies. 7,11 Following all interventions, the rats were euthanized with pentobarbital sodium (0.5 mg/kg body weight, i.p.; Kyoritsu Seiyaku), and the cerebellum was removed. HFD-ET and HFD-ETMT rats were euthanized at least 36 hours after the last exercise session.
All experiments were approved by the Animal-Care Committee of Doshisha University.

| Immunoblotting analysis
The cerebellum was homogenized in ice-cold EzRIPA lysis buffer (ATTO). The homogenate was centrifuged twice for 20 minutes at 14 000 g at 4°C; the total protein concentration in the supernatant obtained was then measured using a BCA protein assay kit (Takara Bio). The same amounts of protein in each sample were run on SDS-PAGE (8%-12.5% gel). After electrophoresis, the proteins were transferred onto a PVDF membrane and blocked for 5 minutes in Bullet Blocking One (Nacalai Tesque) or for 60 minutes with Trisbuffered saline (20 mmol/L Tris, 0.15 mol/L NaCl, pH 7.4) containing 0.1% Tween-20 and 5% skimmed milk. Membranes were incubated overnight at 4°C with a 1:1000 dilution of specific antibodies: BDNF, TrkB, GAPDH (Abcam); cAMP response element binding protein (CREB) and phospho-CREB (CST Japan). The membranes were labeled for 60 minutes with anti-rabbit or anti-mouse immunoglobulin G (1:2500; GE Healthcare). Bands were visualized using the ECL system (GE Healthcare) and quantified on the ChemiDocTM MP system (Bio-Rad). Protein abundance was normalized to GAPDH.

| Cytokine array analysis
Cytokine array analysis was performed using the Rat Cytokine Antibody Array (Abcam) according to the manufacturer instruction.
Protein samples from individual rats were pooled to ensure equal volumes in each experimental group. This compensated for lower volume samples and could mitigate the effects of biological sample variation. Images were acquired using the ChemiDocTM MP system (Bio-Rad), and the pixel intensity was quantified using Image J (National Institutes of Health).

| Statistical analysis
All data, except the cytokine array analysis data, are presented as means ± SE and were analyzed by one-way analysis of variance.
Where applicable, the Bonferroni test for multiple comparisons was conducted. A P-value < .05 or less following post hoc analysis was considered significant. All analyses were performed using the Excel software package.

| D ISCUSS I ON
Recent advances suggest that obesity and HFD feeding deteriorates cognitive function via abnormalities of BDNF levels. 1-3 However, this report did not observe HFD-induced reduction of cerebellar BDNF. It appears valid to conclude that the effect of HFD on brain BDNF levels remains unclear; some studies reported that HFD reduced BDNF levels in the hippocampus 12-14 and cerebral cortex 15 but had no effect on hippocampal BDNF. 16,17 Furthermore, HFD-ET and HFD-MT rats did not exhibit enhanced cerebellar BDNF expression. In this regard, a site-specific difference of BDNF expression may exist between the hippocampus and cerebellum in response to ET. Second, the effect of ET on BDNF expression may be considered as an acute effect rather than a chronic one 18 ; the increased hippocampal BDNF expression was found at 2 hours, but not 2 days after 4 weeks of ET. 5 We collected the cerebellum 36 hours after the last exercise session. Finally, the doses of melatonin were probably inadequate for increasing BDNF expression; a report suggests that even at 40 mg/kg for 21 days, melatonin increased hippocampal BDNF levels by only 17% compared with controls. 19 Even under these conditions, ET combined with MT demon- and thiobarbituric acid reactive substances. 27 Melatonin may potentiate the effect of ET through its antioxidant and anti-inflammatory properties. 28 Brain-derived neurotrophic factor and inflammatory cytokines are believed to affect the expression/function of each other; BDNF downregulates TNF-α expression and upregulates IL-10 expression 29 ; IL-1β upregulates or decreases both, hippocampal BDNF and TrkB expression following single or chronic injections, respectively. 30 However, such an orchestrated change F I G U R E 2 Heat map of cytokine expression in the cerebellum following 9 wk of intervention. The relative expression levels (fold change) of each cytokine were determined by comparing the designated protein concentration of each sample relative to the median value of the designated cytokine across all samples. A, Heat map for all 34 cytokines, B, The cytokines displaying greater than twofold changes were selected in HFD-SED rats. The images were shown in Figure S1. The color red indicates relatively high protein expression, and the color blue indicates relatively low protein expression in BDNF and inflammatory cytokine expression was not always found; although the expression profiles of cytokines were quite different between all groups, elevated BDNF levels were found in HFD-MTET rats only. More detailed data will be needed before establishing the functional interaction of BDNF expression with inflammatory cytokines.
In conclusion, our data suggest that ET combined with MT may play a potential role in elevating BDNF and improving HFD-induced dysregulations of cerebellar cytokines.

ACK N OWLED G M ENTS
This study was supported in part by Private University Research Branding Project; Ministry of Education, Culture, Sports, Science and Technology (19H04010 to TI, 17K19936 to H.T, 18K17874 to HK).

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interest.

AUTH O R CO NTR I B UTI O N S
AI, HT, and TI conceived and designed the study. AI performed most of the experiments with assistance from HK, SO, YM, and HT. AI and TI analyzed the data and wrote the manuscript. TI edited the manuscript.

A N I M A L S TU D I E S
All animal experiments were approved by the Animal-Care Committee of Doshisha University.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available in the Data S1 of this article.