Voluntary exercise suppresses inflammation and improves insulin resistance in the arcuate nucleus and ventral tegmental area in mice on a high-fat diet

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Introduction
Obesity is a growing global public health problem [1,2].Obesity leads to an increased risk of diseases such as hypertension, dyslipidemia, type 2 diabetes mellitus, fatty liver, stroke, coronary artery disease, sleep apnea syndrome, and several cancers [3,4].In recent years, lifestyle factors, such as decreased physical activity and the consumption of fat-rich fast food, have been identified as causes of obesity [5].
In the pathogenesis of obesity under a high-fat diet (HFD) conditions, we and other research groups have shown that hypothalamic inflammation with microglial activation due to HFD precedes substantial weight gain in mice [6][7][8].Hypothalamic inflammation causes insulin and leptin resistance in the hypothalamus resulting in obesity due to increased food intake and decreased energy expenditure [9].In addition, hypothalamic inflammation impairs glucose metabolism independently of weight changes [10].
Another aspect of the mechanism of obesity development under HFD conditions is that HFD induces abnormal feeding behavior [11].In addition to homeostatic system in the hypothalamus, reward system whereby dopaminergic neurons in the ventral tegmental area (VTA) project to the nucleus accumbens also regulates feeding behavior [12].Palatable food, such as HFD, adversely affects the functioning of the reward system leading to obesity [13].We have previously shown that HFD induced inflammation as shown by activated microglia and insulin resistance in the VTA [14].Using mice that have a deficiency of the inhibitor of nuclear factor-Kβ kinase (IKKβ) specifically in dopaminergic neurons, we also showed that an improvement in insulin resistance due to decrease of HFD-induced inflammation in the VTA ameliorated binge-like feeding behavior when on HFD [15].Since insulin exerts its effect on the hypothalamus to inhibit weight gain [16] and on the VTA to suppress hedonic feeding [17], improving inflammation-derived insulin resistance in both areas that are induced by HFD is critical from the perspective of the treatment of obesity.
Exercise is as important as diet in the treatment of obesity [18,19].It is reported that not only forced exercise by treadmill but also voluntary wheel running was effective for preventing obesity in mice [20].Several lines of evidence exist showing that voluntary wheel running reduces weight gain [21,22], and improves glucose intolerance [23,24] and insulin resistance [25] under HFD conditions.Voluntary exercise improves insulin resistance [26] and suppresses microglial activation in the hypothalamus [27].However, the effects of voluntary exercise on HFD-induced inflammation in the hypothalamus, especially the direct effect of the exercise independent of weight differences, remain unclear.It also remains to be clarified how inflammation and the insulin resistance that develops in the VTA under HFD conditions can be affected by exercise.
In the present study, we investigated the effects of voluntary exercise, using a running wheel, on HFD-induced inflammation and insulin resistance in the arcuate nucleus (ARC) and VTA, together with changes in glucose and energy metabolism.

Mice
We used C57BL6J male mice (Japan SLC, Shizuoka, Japan) in this study.They were fed either a chow diet (CHD) (CE-2; the proportions of calories provided by carbohydrate, protein, and fat were 70.5%, 24.9%, and 4.6% respectively; CLEA Japan, Tokyo, Japan) or HFD (58Y1; the proportions of calories provided by carbohydrate, protein, and fat were 20.1%, 18.3%, and 60.9% respectively; TestDiet, Richmond, IN, USA).The Animal Care and Use Committee of Nagoya University Graduate School of Medicine approved all procedures performed in animal experiments (approval number: M230079, approval date: February 14, 2023) and performed according to the US National Institutes of Health animal care guidelines.Mice lived under a 12 h light/12 h dark cycle (lights on from 9 am to 9 pm) in a temperature-controlled barrier environment with ad libitum access to food and water.

Experimental design
First, we divided 8-week-old mice into two groups: sedentary or exercise groups.Mice in the sedentary group were housed in cages without running wheels.Mice in the exercise group were housed in cages with running wheels that could be freely accessed throughout the experiment.In the exercise group, animals underwent two days of acclimatization while on CHD.After acclimatization, half of each of the sedentary and exercise groups were fed CHD and the other half of the two groups were fed HFD.Thus, we divided mice into four groups: CHDfed sedentary group (CHD/no exercise [EX-] group), HFD-fed sedentary group (HFD/EX-group), CHD-fed exercise (EX+) group (CHD/EX+ group), and HFD-fed exercise group (HFD/EX+ group).After four weeks of experimentation, all mice were sacrificed and brains, and adipose and muscle tissues were collected for further analysis (Supplemental Fig. 1).To confirm that the anti-inflammatory effects of exercise were independent of body weight differences, HFD was administered to 8-weekold HFD/EX-and 9-week-old HFD/EX+ mice for 3 days, respectively, and the analysis of mRNA expression in the ARC and VTA was performed after dissection.The reason for comparing HFD/EX-and HFD/EX+ mice at different ages is that significant differences occurred in body weight 3 days after HFD administration when compared at the same age (data not shown).The mice in CHD/EX-and CHD/EX+ groups were 9-week-old mice used as controls, and no weight differences were observed between CHD/EX-, CHD/EX+, HFD/EX-, and HFD/EX+ mice at dissection.

Measurement of factors related to energy metabolism
Body weight was measured weekly.Food intake was assessed weekly by multifeeders (Shinfactory, Fukuoka, Japan) and cumulative food intake was calculated for four weeks.Feed efficiency was calculated as grams of body weight gained per calories of food consumed during the last week of each experiment.After four weeks of experimentation, the rectal temperature was measured using a Heating Pad System for Rodents (FHC-HPS; Muromachi Kikai, Tokyo, Japan), and the weights of epididymal fat pads, brown adipose tissue, and soleus and gastrocnemius muscles were determined.

Exercise
We used wireless running wheels (ENV-047; Med Associates, Fairfax, VA, USA) for voluntary exercise.The revolutions of the wheels were recorded every 30 sec and the moving distances were calculated automatically by dedicated software (SOF-860; Med Associates).We calculated the total moving distances during each week.The duration of the exercise was set at four weeks, based on a previous report [28].

Glucose and insulin tolerance test
A glucose tolerance test (GTT) was conducted on mice that had been fasting for 8 h following four weeks of experimentation as described previously [29].Plasma insulin levels were determined at 0 and 30 min as described previously [29].An insulin tolerance test (ITT) was also performed as described previously [29].The insulin dose was 0.75 or 1.0 mU/g body weight for the ITT and blood glucose levels were assayed as described previously [29].

Hyperinsulinemic-euglycemic clamp
We used a hyperinsulinemic-euglycemic clamp (HEC) to assess the insulin resistance of mice in the HFD/EX-and HFD/EX+ groups after four weeks of experimentation.We used the HEC as described previously [30].The ARC and VTA were dissected from mice after four weeks of experimentation.Before dissection, mice were anesthetized and then transcardially perfused with phosphate buffered saline.The ARC and VTA were then dissected as described previously [8,14].The dissected ARC and VTA were stored at -80 • C. Total RNA was extracted from the samples and cDNA was synthesized from 200-300 ng of total RNA.Quantitative real-time polymerase chain reaction (qRT-PCR) was then performed as described previously [14].The relative mRNA levels of tumor necrosis factor alpha (TNFα), interleukin-6 (IL6), interleukin-1 beta (IL1β), ionized calcium-binding adaptor molecule 1 (Iba1), interleukin-10 (IL10), and cluster of differentiation molecule 80 (CD80) were determined using qRT-PCR and glyceraldehyde 3-phosphate dehydrogenase as an internal control.The comparative Ct method was used to calculate relative mRNA expression levels as described previously [8].Primer sequences are described in Table S1.

Intracerebroventricular injection of insulin
After 8 h of fasting, we injected insulin into the lateral ventricles of mice as described previously [14] after four weeks of experimentation.The concentration of insulin was 10⁻⁵ M. We dissected the ARC and VTA 15 min after insulin injection and stored samples at -80 • C.

Statistical analysis
We conducted data analysis using the SPSS Statistics 28 software (IBM, NYC, NY, USA).To assess the statistical significance of group differences, we employed either unpaired t-tests, one-or two-way analysis of variance (ANOVA) with repeated measures, or two-way factorial ANOVA, followed by Bonferroni post hoc tests as deemed suitable.The findings are presented as mean ± standard error of the mean (SEM), and statistical significance was established at p < 0.05.

Voluntary wheel running suppresses weight gain under HFD conditions
Body weight change was calculated as the sum of body weights gained per week from the beginning of the experiment (Fig. 1A).The original weights among the four groups are shown in Supplemental Fig. 2. Body weight change in mice of the HFD/EX-group was significantly increased compared to those of the CHD/EX-and HFD/EX+ groups.No significant differences were found in body weight change between mice of the CHD/EX-, CHD/EX+ and HFD/EX+ groups (Fig. 1A).Cumulative food intake by mice in the CHD/EX+ and HFD/ EX-groups was significantly higher compared to that in the CHD/EXgroup.No significant difference was found in the cumulative food intake between HFD/EX-and HFD/EX+ groups (Fig. 1B).While food intake in the light cycle in the HFD/EX-group was similar to that in the dark cycle, food intake in the light cycle was significantly less than in the dark cycle the HFD/EX+ group (Supplemental Fig. 2B).Feed efficiency in the HFD/EX-group was significantly increased compared to that in the CHD/EX-group.The feed efficiency in the HFD/EX+ group was significantly decreased compared to that in the HFD/EX-group (Fig. 1C).Wheel running distance by mice in the HFD/EX+ group was significantly longer than that in the CHD/EX+ group (Fig. 1D).The wheel running distance in the HFD/EX+ group was significantly increased compared to the CHD/EX+ group in the dark cycle, but not in the light cycle (Supplemental Fig. 2C).Rectal temperature in the CHD/ EX+ group was significantly higher than that in the CHD/EX-group (Fig. 1E).The weights of epididymal fat pads and brown adipose tissue were significantly increased in mice of the HFD/EX-compared to CHD/ EX-group.Adipose tissue weights were significantly decreased in mice of the HFD/EX+ compared to HFD/EX-group (Fig. 1F and G).No significant differences were found in the weights of soleus and gastrocnemius muscles between mice of the four groups (Fig. 1H and I).

Voluntary wheel running improves impaired glucose metabolism under HFD conditions
With regard to the GTT, blood glucose (BG) levels in mice of the HFD/EX-group were significantly higher than those in mice of the CHD/ EX-group at each time point (Fig. 2A).The BG levels of mice in the HFD/ EX+ group were significantly lower compared to those of mice in the HFD/EX-group at 15, 60, 90, and 120 min; however, these were higher than those of mice in the CHD/EX+ group at 60, 90, and 120 min (Fig. 2A).No significant differences were noted at each time point between mice of CHD/EX-and CHD/EX+ groups (Fig. 2A).The area under the curve (AUC) of the GTT for mice in the HFD/EX-group was significantly greater than that of mice of the CHD/EX-group.The AUC of the GTT for mice of the HFD/EX+ group was significantly lower than that of mice of the HFD/EX-group but still higher than that of mice of the CHD/ EX+ group (Fig. 2A).Plasma insulin levels 0 and 30 min after glucose injections in mice of the HFD/EX-group were significantly higher than those of mice in the CHD/EX-group.Plasma insulin levels in mice of the HFD/EX+ group were significantly lower than those of mice in the HFD/ EX-group (Fig. 2B and 2C).The homeostatic model assessment for insulin resistance (HOMA-IR), an index of insulin resistance [31,32] was calculated as follows: fasting insulin (μU/mL) × fasting glucose (mmol/L)/22.5.The insulinogenic index was calculated as follows: change in insulin divided by change in glucose during the first 30 min of the GTT [33].Both indices in mice of the HFD/EX-group were significantly higher than those of mice in the CHD/EX-group (Fig. 2D and E).The HOMA-IR of mice of the HFD/EX+ group was significantly lower than that of mice of the HFD/EX-group, but still higher than that of mice of the CHD/EX+ group (Fig. 2D).With regard to ITT, BG levels in mice of the HFD/EX-group were significantly higher than those of mice in the CHD/EX-group at each time point (Fig. 2F).The BG levels in mice of the  S2.ANOVA: analysis of variance, CHD: a chow diet, EX+: exercise, EX-: no exercise, HFD: a high-fat diet, SEM: standard error of the mean.HFD/EX+ group were significantly lower than those of mice in the HFD/EX-group at 45 min, but still higher than those of mice in the CHD/EX+ group at each time point except for 120 min (Fig. 2F).The BG levels in mice of the CHD/EX+ group were significantly lower than those of mice in the CHD/EX-group at 15, 30, and 45 min (Fig. 2F).The AUC of ITT for mice of the HFD/EX-group was higher than that of mice in the CHD/EX-group (Fig. 2F).The AUC of ITT for mice in the HFD/EX+ group had a tendency to be decreased compared to that of mice in the HFD/EX-group, but did not reach a significant difference (Fig. 2F).The AUC of ITT in mice of the CHD/EX+ group was significantly lower than those of mice in CHD/EX-and HFD/EX+ groups (Fig. 2F).With respect to the hyperinsulinemic-euglycemic clamp, the glucose infusion rate (GIR) for mice in the HFD/EX+ group was significantly higher than that of mice in the HFD/EX-group (Fig. 2G).Since 0.75 U/kg of insulin was not considered a sufficient dose to determine the difference in the AUC of ITT between HFD/EX-and HFD/EX+ groups, an additional experiment was conducted at 1.0 U/kg.The AUC of ITT using 1.0 U/kg insulin for mice in the HFD/EX+ group was significantly lower than that of mice in the HFD/EX-group (Supplemental Fig. 3).

Analysis of changes in mRNA expression of inflammation-related cytokines and microglial cell markers in the ARC
In the ARC, we found that mRNA expression of the inflammatory cytokines, TNFα, IL6, and IL1β, were significantly increased in the tissues of mice in the HFD/EX-compared to CHD/EX-group (Fig. 3).Of note, the mRNA expression of TNFα in mice of the HFD/EX+ group was significantly lower than that of mice in the HFD/EX-group (Fig. 3).The mRNA expression of IL6 in mice of the CHD/EX+ group was significantly higher than that of mice in the CHD/EX-group; no significant difference was found for the anti-inflammatory cytokine, IL10, between the four groups (Fig. 3).The mRNA expression of the microglial cell markers, Iba1 and CD80, in the ARC were significantly increased in mice of the HFD/EX-compared to CHD/EX-group (Fig. 3).Interestingly, the mRNA expression of CD80, a microglial M1 marker, in the tissues of mice in the HFD/EX+ group was significantly lower than that in tissues of mice in the HFD/EX-group (Fig. 3).

Immunohistochemical analysis of changes in expression of microglial TNFα in the ARC
The immunohistochemical analysis of the ARC was performed for the four mice groups.Microglia that were TNFα-positive were observed in the ARC of mice in HFD groups (Fig. 4A).For a more accurate assessment, the percentage of TNFα-positive microglia among total microglia inside the ARC was calculated of each group (Fig. 4B).The proportion of TNFα-positive cells in the ARC of mice in the HFD/EX-group was significantly greater than that for mice in the CHD/EX-group (Fig. 4B).Of note, the proportion of TNFα-positive cells in mice of the HFD/EX+ group was significantly lower than that in mice of the HFD/EX-group, but still higher than that in mice of the CHD/EX+ group (Fig. 4B).

Analysis of changes in mRNA expression of inflammatory-related cytokines and microglial cell markers in the VTA
The mRNA expression of the inflammatory-related cytokines, TNFα and IL10, were significantly increased in the VTA of mice in the HFD/ EX-compared to CHD/EX-group (Fig. 5).Of note, the mRNA expression of TNFα in mice of the HFD/EX+ group was significantly lower than that in mice of the HFD/EX-group (Fig. 5).Significant differences in the inflammatory cytokines, IL6 and IL1β, in the VTA was not found between mice of the four groups (Fig. 5).The mRNA expression of the microglial cell markers, Iba1 and CD80, in the VTA were significantly increased in mice of the HFD/EX-compared to CHD/EX-group (Fig. 5).Interestingly, the mRNA expression of CD80, a microglial M1 marker, in mice of the HFD/EX+ group was significantly lower than that in mice of the HFD/EX-group (Fig. 5).

Immunohistochemical analysis of the expression changes of microglial TNFα in the VTA
Immunohistochemical analysis of the VTA was performed for the four groups.Microglia that were TNFα-positive were noted in mice of the HFD groups (Fig. 6A).For a more accurate assessment, the percentage of TNFα-positive microglia among total microglia was calculated inside the VTA of mice in each group (Fig. 6B).The proportion of TNFα-positive cells in the VTA of mice in the HFD/EX-group was significantly higher than that in mice of the CHD/EX-group and was Fig. 3. Voluntary wheel running suppresses HFD-induced inflammation in the ARC.The mRNA expression levels of inflammation-related cytokines and glial cell markers in the ARC.All values are mean ± SEM.The differences between groups were analyzed for statistical significance by two-way factorial ANOVA and then a Bonferroni post hoc test.#p < 0.05, vs. CHD/EX-; †p < 0.05, vs. CHD/EX-; *p < 0.05, vs. HFD/EX-.The details of statistics are shown in Table S2.ANOVA: analysis of variance, ARC: arcuate nucleus, CHD: a chow diet, EX+: exercise, EX-: no exercise, HFD: a high-fat diet, SEM: standard error of the mean.significantly lower in the VTA of mice in the CHD/EX+ group compared to that of mice in the CHD/EX-group (Fig. 6B).Of note, the proportion of TNFα-positive cells in the VTA of mice in HFD/EX+ group was significantly lower than that in the VTA of mice in the HFD/EX-group, but still higher than that in the VTA of mice in the CHD/EX+ group (Fig. 6B).

Voluntary wheel running improves HFD-induced insulin resistance in the ARC and VTA
In the ARC and VTA, the phosphorylation of Akt after central insulin injection in mice of the HFD/EX-group was significantly lower than that in mice of the CHD/EX-group.The phosphorylation of Akt in the ARC and VTA of mice in the HFD/EX+ group was significantly higher than that in mice of the HFD/EX-group (Fig. 7A and B).S2.ANOVA: analysis of variance, CHD: a chow diet, EX+: exercise, EX-: no exercise, HFD: a high-fat diet, SEM: standard error of the mean, VTA: ventral tegmental area.

Voluntary exercise suppresses inflammation in the ARC independently of weight changes under HFD conditions
The inflammation in the ARC occurs prior to substantial body weight gain early after HFD administration, and is considered a cause of obesity associated with HFD ingestion [6][7][8]34,35].To elucidate whether voluntary exercise suppresses hypothalamic inflammation induced by HFD independently of body weight changes, mice with no weight difference between the four groups on day 3 after HFD or chow administration were selected to evaluate ARC inflammation.mRNA expression of the inflammatory cytokine, TNFα, in the ARC was significantly increased in mice of the HFD/EX-compared to CHD/EX-group   S2.ANOVA: analysis of variance, ARC: arcuate nucleus, CHD: a chow diet, EX+: exercise, EX-: no exercise, HFD: a high-fat diet, SEM: standard error of the mean, VTA: ventral tegmental area.
(Fig. 8A).Of note, the mRNA expression of TNFα in mice of the HFD/EX+ group was significantly lower than that in mice of the HFD/EX-group in the ARC (Fig. 8A).The mRNA expression of the inflammatory cytokine, IL6, in the ARC of mice in the CHD/EX+ group was significantly increased compared to that in the ARC of mice in the CHD/EX-group (Fig. 8A).The mRNA expression of the anti-inflammatory cytokine, IL10, in the ARC of mice in the HFD/EX+ group was significantly higher than that in the ARC of mice in the HFD/EX-group (Fig. 8A).The mRNA expression of a microglial activation marker, Iba1, in the ARC was significantly increased in mice of the HFD/EX-compared to CHD/EX-group (Fig. 8A).Interestingly, the mRNA expression of Iba1 in the ARC of mice in the HFD/EX+ group was significantly lower than that in mice of the HFD/EX-group (Fig. 8A).No significant differences were observed in the inflammatory cytokine, IL1β, and a microglial M1 marker, CD80, in the ARC of mice between the four groups (Fig. 8A).
Since inflammation occurred in the VTA 28 days after HFD administration was suppressed by voluntary exercise, we evaluated VTA inflammation on day 3 after HFD or CHD administration between the four groups of mice that did not differ in body weight, as in the ARC experiment.In contrast to the data obtained in the ARC, no significant differences were noted in inflammatory-related cytokines and microglial cell markers of the VTA of mice of the four groups (Fig. 8B).

Discussion
We found in the present study that voluntary wheel running (i) suppressed HFD-induced inflammation in the ARC and VTA with a decrease in microglial activity, especially in the ARC and independently of weight changes; (ii) improved insulin resistance in the ARC and VTA when mice were on HFD; (iii) suppressed HFD-induced obesity; and (iv) improved glucose metabolism in the periphery when mice were on HFD.These findings suggest new insights in that the suppression of central inflammation by voluntary wheel running when mice are on HFD is involved in anti-obesity effects and improvements in glucose metabolism.
The ARC is a fundamental region in the brain regulating energy and glucose metabolism [36,37].Hypothalamic inflammation is known to impair the homeostatic system of energy metabolism by inhibiting insulin and leptin receptor signaling [9,38].In addition, previous reports showed that inhibition of IKKβ/NF-kappaB signaling in the brain, hypothalamus or astrocytes suppressed HFD-induced inflammation to prevent obesity [39,40].We and others have reported that microglia play a pivotal role in provoking HFD-induced inflammation [8,34].By using magnetic-activated cell sorting analysis, we have also previously shown that HFD causes activation of microglia with increased mRNA expression of the M1 marker resulting in hypothalamic inflammation [7].In the present study, we showed that voluntary wheel running reduced an increase in the mRNA expression of TNFα, and the percentage of TNFα and microglial co-stained by immunohistochemical analysis in the ARC.In addition, we also showed that voluntary running reduced the mRNA expression of CD80, a microglial M1 marker [41], suggesting a decrease in microglial activity on HFD.Furthermore, the suppression of both inflammation and microglial activity in the ARC is observed in mice of the exercise group as early as 3 days after HFD administration, independently of body weight changes.Overall, these findings suggest that exercise may contribute to the suppression of body weight gain by reducing the inflammation associated with HFD administration from an early stage, and are considered to be extremely important from the therapeutic perspective of obesity induced by HFD.One possible mechanism for the suppression of inflammation in the ARC is that voluntary exercise-induced myokines suppressed microglial activity when mice were on HFD since myokines, such as IL4, IL6, and IL10, can cross the blood-brain barrier [42] and inhibit microglial activity [43].In the present study, consistent with a previous report [44], the mRNA expression of IL6 in the ARC of mice in the CHD/EX+ group was significantly increased compared to that in mice of the CHD/EXgroup.The IL6 expression in the ARC had a tendency to increase in mice of the HFD/EX+ group compared to animals in the HFD/EX-group after 28 days of HFD but the difference did not reach statistical significance.As for the relationship between muscle myokines and brain function, IL6 may contribute to suppress HFD-induced inflammation to some content by inhibiting microglial activation in the ARC.
Previous studies showed that hypothalamic inflammation caused insulin resistance in the hypothalamus, which led to obesity due to increased food intake and decreased energy expenditure [9].In the present study, it was shown that insulin resistance in the ARC was improved with the suppression of HFD-induced inflammation by voluntary exercise.Considering previous reports that showed the suppression of inflammation improved insulin resistance in the hypothalamus leading to improved glucose and energy metabolism [39], it is possible that the suppression of HFD-induced inflammation by voluntary exercise contributed to the improvement in glucose and energy metabolism under HFD conditions.
The VTA is one of the main components of the brain's reward system [45,46].The activation of neurocircuitry in the reward system by  S2.ANOVA: analysis of variance, ARC, arcuate nucleus; CHD: a chow diet, EX+: exercise, EX-: no exercise, HFD: a high-fat diet, SEM: standard error of the mean, VTA: ventral tegmental area.
palatable food consumption, such as in an HFD, causes hedonic feeding behavior, even when nutritional supplementation is not needed [47].In addition, highly palatable foods also induce binge eating, which may lead to obesity [48].By contrast, insulin inhibits overeating and hedonic eating behavior by binding to insulin receptors expressed on dopaminergic neurons in the VTA [17].We have previously shown that HFD led to inflammation and insulin resistance in the VTA, as shown by increased mRNA expression of Iba1, increased TNFα expression, and decreased phosphorylation of Akt in the VTA, all of which suggest microglial activation [14].We also showed that in mice with a deficiency of IKKβ, specifically in dopaminergic neurons, the suppression of inflammation in the VTA improves abnormal eating that is associated with insulin resistance [15].In the present study, similar to data obtained in the ARC, voluntary wheel running suppressed HFD-induced CD80 and TNFα expression in the VTA.In addition, our data showed that voluntary exercise suppressed HFD-induced inflammation and improved insulin resistance in the VTA, as well as in the ARC.The mechanism of suppression of inflammation in the VTA is considered to be basically similar to that in the ARC.Regarding insulin receptor signaling, voluntary exercise is well known to improve insulin resistance at the periphery [25,49].However, the findings of the present study demonstrate that exercise improves insulin resistance not only in the periphery but also in the center, such as in the ARC and VTA, consistent with previous studies [50,51].
Our data showed that in the HFD group, voluntary wheel running suppressed weight gain to the same level as in the CHD group.Although food intake was not significantly different between exercise and no exercise groups on HFD, weight gain was significantly reduced by exercise.Consistent with a previous paper [52], food intake in the light cycle was similar to that in the dark cycle in the HFD/EX-group, while food intake in the light cycle was lower than in the dark cycle in the HFD/EX+ group.These data suggest that exercise restored the disrupted rhythm of food consumption induced by an HFD, at least in part.Feed efficiency, an index of energy expenditure, was increased in the HFD group but decreased by exercise, which is consistent with a previous report [53], suggesting that the mechanism of suppression of body weight gain is due to an increase in energy expenditure.This increase in energy expenditure is possibly related to the suppression of ARC inflammation by exercise [40,54].In addition, the energy expenditure of voluntary exercise using a wheel was 1.2 kJ/km [55], suggesting that energy expenditure by exercise itself also contributed to the suppression of body weight gain.In terms of body composition, no change in muscle weight occurred, and decreases in white and brown fat weights were observed, suggesting that the suppression of fat gain contributed to the suppression of body weight gain in the exercise group on HFD.There are two major types of running wheels used in mouse studies: upright wheels and angled wheels [28], the latter of which was employed in the present study.Running wheel design may affect total voluntary running activity in mice, with higher voluntary running activity associated with angled wheels compared with upright wheels [28].However, the effects of voluntary exercise with angled wheels on body weight under HFD and CHD conditions in the present study were similar to those of previous papers that used upright wheels [56,57].It should be noted that activities other than wheel running were not measured in our experiment and will be the subject of future studies.
Our data also showed that voluntary wheel running improved impaired glucose tolerance and insulin resistance in the periphery when mice were on HFD.The suppression of hypothalamic inflammation improves glucose tolerance independently of changes in body weight [58,59] and that exercise increases glucose uptake into skeletal muscle [60].Overall, the improvement in glucose metabolism observed in the exercise group on HFD in the present study was considered to be due to the combined effects of an exercise-induced reduction of fat mass, an increase in glucose uptake in skeletal muscle, and the suppression of hypothalamic inflammation.Of note, voluntary exercise with angled wheels during a 4-week HFD suppressed weight gain to the same level as in the CHD group; however, it did not improve glucose tolerance or insulin sensitivity to the same level as in the CHD group.These results are consistent with previous studies that used upright wheels [56,57].This may be because dietary modification is more important for improving glucose metabolism than voluntary exercise [57].
In the present study, it remains unclear whether the mechanism by which voluntary exercise improves inflammation in the ARC and VTA when mice are on HFD is due to a direct effect of exercise or a secondary effect due to a reduction in fat mass.At least for the ARC, the suppression of inflammation by exercise was independent of weight changes, suggesting that both direct and secondary effects were involved.It also remains unclear which specific myokines produced during voluntary exercise are effective in suppressing microglial activation in the ARC and VTA under HFD conditions.Thus additional studies are necessary to elucidate detailed mechanisms in the future.
In conclusion, we demonstrated that voluntary wheel running ameliorated HFD-induced inflammation in the ARC and VTA with a reduction in microglial activity in mice.The HFD-induced insulin resistance in the ARC and VTA was also improved by voluntary running.Our data indicate that the actions of voluntary exercise on the central nervous system are effective against obesity and impaired glucose metabolism induced by HFD.This provides a possible elucidation of the mechanism by which voluntary exercise suppresses obesity and improves glucose metabolism.

Fig. 2 .
Fig. 2. Voluntary wheel running improves impaired glucose metabolism under HFD conditions.(A) The GTT and AUC) of the GTT, (B and C) plasma insulin levels at 0 and 30 min after glucose injection during the GTT, (D) HOMA-IR, (E) the insulinogenic index, (F) the ITT and the AUC of the ITT, and (G) the GIR during a hyperinsulinemic-euglycemic clamp.All values are mean ± SEM.The differences between groups were analyzed for statistical significance by two-way ANOVA with repeated measures (GTT in A and ITT in F), two-way factorial ANOVA (AUC in A, B-E, and AUC in F) and then a Bonferroni post hoc test and unpaired t-test (G).#p < 0.05, vs. CHD/EX-; †p < 0.05, vs. CHD/EX-; *p < 0.05, vs. HFD/EX-; ‡p < 0.05, vs. CHD/EX+.The details of statistics are shown in TableS2.ANOVA: analysis of variance, AUC: area under the curve, CHD: a chow diet, EX+: exercise, EX-: no exercise, HFD: a high-fat diet, GIR: glucose infusion rate, GTT: glucose tolerance test, HOMA-IR: homeostatic model assessment for insulin resistance, ITT: insulin tolerance test, SEM, standard error of the mean.

Fig. 4 .
Fig. 4. Voluntary wheel running suppresses HFD-induced inflammation in the ARC.(A) Immunostaining of Iba1 (red) and TNFα (green) in the ARC.White arrowheads show the colocalization of TNFα with microglia.(B) The proportion of TNFα-positive microglia.All values are mean ± SEM and scale bar = 20 µm.The differences between groups were analyzed for statistical significance by two-way factorial ANOVA and then a Bonferroni post hoc test.#p < 0.05, vs. CHD/EX-; *p < 0.05, vs. HFD/EX-; ‡p < 0.05, vs. CHD/EX+.The details of statistics are shown in TableS2.ANOVA: analysis of variance, ARC: arcuate nucleus, CHD: a chow diet, EX+: exercise, EX-: no exercise, HFD: a high-fat diet, Iba1, ionized calcium binding adapter molecule 1, SEM: standard error of the mean, TNFα: tumor necrosis factor alpha.

Fig. 5 .
Fig. 5. Voluntary wheel running suppresses HFD-induced inflammation in the VTA.The mRNA expression levels of inflammation-related cytokines and glial cell markers in the VTA.All values are mean ± SEM.The differences between groups were analyzed for statistical significance by two-way factorial ANOVA and then a Bonferroni post hoc test.#p < 0.05, vs. CHD/EX-; *p < 0.05, vs. HFD/EX-.The details of statistics are shown in TableS2.ANOVA: analysis of variance, CHD: a chow diet, EX+: exercise, EX-: no exercise, HFD: a high-fat diet, SEM: standard error of the mean, VTA: ventral tegmental area.

Fig. 7 .
Fig. 7. Voluntary wheel running improves HFD-induced insulin resistance in the ARC and VTA.(A and B) Phosphorylation of Akt 15 min after insulin injection into the third ventricle in ARC (A) and VTA (B).All values are mean ± SEM.The differences between groups were analyzed for statistical differences by two-way factorial ANOVA and then a Bonferroni post hoc test.#p < 0.05, vs. CHD/EX-; *p < 0.05, vs. HFD/EX-.The details of statistics are shown in TableS2.ANOVA: analysis of variance, ARC: arcuate nucleus, CHD: a chow diet, EX+: exercise, EX-: no exercise, HFD: a high-fat diet, SEM: standard error of the mean, VTA: ventral tegmental area.

Fig. 8 .
Fig. 8. Voluntary wheel running suppresses HFD-induced inflammation in the ARC in-dependently of a reduction in body weight.(A and B) The mRNA expression levels of inflammation-related cytokines and glial cell markers in the ARC (A) and VTA (B).All values are mean ± SEM.The differences between groups were analyzed for statistical differences by two-way factorial ANOVA and then a Bonferroni post hoc test.#p < 0.05, vs. CHD/EX-; †p < 0.05, vs. CHD/EX-; *p < 0.05, vs HFD/EX-.The details of statistics are shown in TableS2.ANOVA: analysis of variance, ARC, arcuate nucleus; CHD: a chow diet, EX+: exercise, EX-: no exercise, HFD: a high-fat diet, SEM: standard error of the mean, VTA: ventral tegmental area.