Leigh et al. Nutrition & Metabolism (2020) 17:71 https://doi.org/10.1186/s12986-020-00492-6 RESEARCH Open Access Treadmill exercise has minimal impact on obesogenic diet-related gut microbiome changes but alters adipose and hypothalamic gene expression in rats Sarah-Jane Leigh1, Nadeem O. Kaakoush1, Rosa M. Escorihuela2, R. Frederick Westbrook3 and Margaret J. Morris1* Abstract Background: Exercise has been extensively utilised as an effective therapy for overweight- and obesity-associated changes that are linked to health complications. Several preclinical rodent studies have shown that treadmill exercise alongside an unhealthy diet improves metabolic health and microbiome composition. Furthermore, chronic exercise has been shown to alter hypothalamic and adipose tissue gene expression in diet-induced obesity. However, limited work has investigated whether treadmill exercise commenced following exposure to an obesogenic diet is sufficient to alter microbiome composition and metabolic health. Methods: To address this gap in the literature, we fed rats a high-fat/high-sugar western-style cafeteria diet and assessed the effects of 4 weeks of treadmill exercise on adiposity, diet-induced gut dysbiosis, as well as hypothalamic and retroperitoneal white adipose tissue gene expression. Forty-eight male Sprague-Dawley rats were allocated to either regular chow or cafeteria diet and after 3 weeks half the rats on each diet were exposed to moderate treadmill exercise for 4 weeks while the remainder were exposed to a stationary treadmill. Results: Microbial species diversity was uniquely reduced in exercising chow-fed rats, while microbiome composition was only changed by cafeteria diet. Despite limited effects of exercise on overall microbiome composition, exercise increased inferred microbial functions involved in metabolism, reduced fat mass, and altered adipose and hypothalamic gene expression. After controlling for diet and exercise, adipose Il6 expression and liver triglyceride concentrations were significantly associated with global microbiome composition. Conclusions: Moderate treadmill exercise induced subtle microbiome composition changes in chow-fed rats but did not overcome the microbiome changes induced by prolonged exposure to cafeteria diet. Predicted metabolic function of the gut microbiome was increased by exercise. The effects of exercise on the microbiome may be modulated by obesity severity. Future work should investigate whether exercise in combination with microbiome-modifying interventions can synergistically reduce diet- and obesity-associated comorbidities. Keywords: Obesity, Microbiome, Exercise, Hypothalamus, White adipose tissue, Cafeteria diet * Correspondence: m.morris@unsw.edu.au 1 Department of Pharmacology, School of Medical Sciences, UNSW, Sydney, NSW 2052, Australia Full list of author information is available at the end of the article © The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Leigh et al. Nutrition & Metabolism (2020) 17:71 Page 2 of 13 Introduction administered with high-fat diet, making it difficult to Overweight and obesity leads to reductions in physical translate the findings to people, in terms of implement- and mental health, and quality of life [1], resulting in in- ing exercise after a history of unhealthy eating and creased direct and indirect costs to the global economy obesity. [2]. Along with gross metabolic changes, obesity is asso- Here we sought to examine whether moderate tread- ciated with altered fecal microbial species diversity [3] mill exercise in rats could exert benefits to gut micro- and composition [4]. Separate studies involving transfer biome composition following exposure to either a of obese human fecal microbiome samples induced fat healthy or a high-fat, high-sugar western-style cafeteria gain in naïve mice [5] and supplementation with diet. We aimed to investigate whether any changes in Akkermansia muciniphila improved insulin sensitivity gut microbiome were associated with altered gene ex- and reduced body weight in overweight and obese pression in white adipose tissue (WAT) and the hypo- people [6], providing some evidence for a potential role thalamus, which are known to be affected by both of diet- and obesity-associated gut microbiota changes in obesogenic diets and exercise. adiposity and metabolic dysfunction. Weight loss through lifestyle intervention is an effect- Materials and methods ive strategy for reducing obesity-related comorbidities Subjects and diet manipulation [7]. One such intervention is moderate exercise, a prac- This protocol was approved by the Animal Care and tical and sustainable approach for people with over- Ethics Committee of UNSW Sydney in accordance with weight and obesity [8]: while exercising at this intensity the Australian guidelines for the use and care of animals is unlikely to cause weight loss independent of caloric for scientific purposes (National Health and Medical restriction, it confers cardiovascular and metabolic bene- Research Council). fits, and assists with weight maintenance [9, 10]. Fur- Forty-eight male Sprague-Dawley rats (6–7 weeks, thermore, regular exercise is known to improve glucose 165-185 g; Animal Resource Centre, Australia) were regulation and insulin sensitivity [11] as well as reducing housed 3/box (18-22 °C; 12 h light/dark) and handled cardiovascular disease [12] and cancer risk [13], and daily for one week while maintained on standard chow there is increasing interest in the effects of exercise on (11 kJ/g; Premium Rat and Mouse Maintenance diet; the gut microbiota. Gordon’s Specialty Stock Feeds, Australia) and water ad The first study to indicate an effect of exercise on fecal libitum. microbiome showed that elite, professional athletes ex- Following acclimatization, weight-matched groups hibited a distinct microbiome composition with in- were randomly allocated to Chow plus water or Cafe- creased microbial species diversity [14]. However, since teria diet (n = 12 rats, n = 4 cages per group) ad libitum athletes consume a distinct diet from healthy people in which comprised 10% sucrose solution alongside the community, further work has been undertaken in ro- commercially-produced cakes, cookies and savoury foods dents to identify the specific effects of exercise on fecal [20] in addition to chow and water. Body weight and 24- microbiome. A recent systematic review of primarily ro- h food intake were measured twice weekly and food in- dent studies concluded that while there was no consist- take was calculated assuming equal intake per rat in ent effect of exercise on microbial species richness, each cage. Body composition was measured during week exercise increases the relative abundance of Firmicutes 7 by EchoMRI-900 (EchoMRI LLC, USA). [15]. The different types of exercise used (forced versus voluntary) have been shown to exert different effects on Treadmill exercise fecal microbiome composition in mice [16, 17] which Following 3 weeks, half the rats in each dietary condition may in part explain the inconsistent findings. were allocated to treadmill exercise (generating 4 Furthermore, there were considerable differences in weight-matched groups: Chow Sedentary (CSed), Chow exercise duration used (6–16 weeks) which may contrib- Exercise (CEx), Cafeteria Sedentary (CafSed) and ute to the range of responses observed. Microbiome Cafeteria Exercise (CafEx)) for 4 weeks until the day be- compositional changes were observed in mice main- fore sacrifice. Moderate exercise consisted of 10-12 m/ tained on a healthy diet following 6 weeks of moderate min for 45 min, five days a week at zero incline. The first treadmill exercise [16] and after 16 weeks in mice fed a week comprised two training sessions (0–10 m/min) high-fat diet [18]. In contrast, eight weeks of low-to- after which time spent at 12 m/min was gradually in- moderate exercise did not confer microbiome creased. Sedentary rats were placed in stationary tread- compositional changes in mice fed a high-fat diet [19]. mills during the exercise protocol. Exercising rats were Furthermore, most studies examining the effects of closely monitored. Three CafEx rats showed signs of fa- treadmill exercise on fecal microbiome in diet-induced tigue, distress or vocalisation, and were removed from obesity used a design where exercise was co- the treadmill and thereafter exercised at a lower Leigh et al. Nutrition & Metabolism (2020) 17:71 Page 3 of 13 intensity (5-6 m/min for 45 min). These rats were ana- Statistical analyses lysed together with moderately exercised CafEx rats. Data were analyzed using two-way between-subjects ANOVA, while measures over time were analysed using 3-way mixed ANOVA. Post-hoc pairwise com- Sample collection parisons were performed using a Tukey adjustment At 7 weeks of diet, rats were deeply anaesthetized (keta- where appropriate (THSD) and presented in the asso- mine/xylazine 15/100 mg/kg intraperitoneally). Body ciated figures and tables only when p < 0.05. Pearson’s weight, naso-anal length, girth and blood glucose were correlations were used to identify associations. All measured following induction of anesthesia. Rats were analyses were completed using IBM SPSS Statistics 23 decapitated for trunk blood. (Australia). The hypothalamus (within coronal block defined by rostro-caudal limits of Circle of Willis) was rapidly dis- sected and collected. Retroperitoneal and gonadal WAT, Fecal DNA extraction, microbiome community sequencing and liver were dissected and weighed. One fecal pellet and statistical analyses was removed from the distal colon. Tissue and feces DNA extraction was performed using the PowerFecal were snap frozen in liquid nitrogen and stored at -80 °C. DNA Isolation Kit (MoBio Laboratories, USA). Micro- bial community composition was assessed by Illumina Protein and triglyceride measurements amplicon sequencing (2 × 250 bp MiSeq chemistry, V4 Plasma leptin and insulin concentrations were measured region, 515F-806R primer pair) using a standard proto- using commercial kits (CAT#90040 and CAT#90060, col. Sequence data were analyzed using Mothur [23] CrystalChem Inc., USA). Plasma and liver triglyceride using modified commands from MiSeq SOP [24], in- content were measured spectrophotometrically using cluding alignment with the SILVA database, singleton triglyceride reagent (Roche Diagnostics Australia Pty removal, chimera checking with UCHIME and classifica- Ltd., Australia) at 37 °C alongside a standard curve tion against the latest RDP training set. Sequence depth generated from glycerol standard (G7793-5ML, Sigma- was normalized by subsampling to 8346 total clean reads Aldrich Pty Ltd., Australia). Livers were extracted by per sample. homogenization in 2:1 chloroform/methanol and incu- Operational taxonomic unit (OTU) correlations bated overnight; 0.6% NaCl was added and samples were were completed using Calypso [25], with the vortexed and centrifuged (1000 g for 10 min). The lower Benjamini-Hochberg false discovery rate (FDR) pro- phase was then evaporated at 40 °C under nitrogen gas. cedure [26] used to control for multiple tests. FDR- Dried extract was re-dissolved in absolute ethanol and corrected DESeq2 was performed using the Phyloseq measured spectrophotometrically. Retroperitoneal WAT [27] R package for the negative binomial Walk test in IL-6 content was determined using a DuoSet IL-6 Rat DESeq2 [28]. OTU abundances were analyzed using ELISA (DY506, R&D Systems Inc.) following manufac- SPSS with Kruskal-Wallis tests, followed by non- turers’ recommendations. parametric Bonferroni-Dunn post-hoc testing where appropriate. OTUs of interest were identified using SINA Aligner [29]. RNA extraction and gene expression assays Alpha diversity metrics, distanced-based linear model- RNA was extracted from hypothalamus and retroperi- ling (dbLM), permutational multivariate ANOVA toneal WAT using TRI Reagent (Sigma-Aldrich Pty Ltd., (PERMANOVA), non-metric multidimensional scaling Australia). Following DNAse I treatment (Catalogue# (nMDS) and canonical analysis of principal coordinates 42885; Merck, Australia), 1.5 or 2 μg of RNA (WAT and were completed using Primer V6 (Primer-E Ltd., hypothalamus respectively) were reverse transcribed to Plymouth, United Kingdom [30]). All Primer analyses produce cDNA (High Capacity Reverse Transcriptase utilized a Bray-Curtis similarity matrix constructed at Kit; Thermofisher Scientific, USA). Gene expression was the OTU level. assessed using Taqman inventoried gene expression as- Phylogenetic Investigation of Communities by Recon- says (Life Technologies Australia Pty Ltd., Australia; see struction of Unobserved States (PICRUSt) was per- Supplementary Table 1). Genes of interest were normal- formed using Galaxy web to predict putative functions ized against the geometric mean of the two most stable (through metagenomic prediction) from the 16S OTU housekeeping genes (Gapdh and Hprt1 for WAT, Hprt1 data using Greengenes 13.5 for taxonomic classification and B2m for hypothalamus) identified by the Normfin- [31]. Pathway counts were compared across groups der package [21]. Analysis of relative gene expression using FDR-corrected Kruskal-Wallis tests followed by was performed using the ΔΔCT method normalized to non-parametric Bonferroni-Dunn post-hoc testing where an independent calibrator [22] made from all samples. appropriate. Leigh et al. Nutrition & Metabolism (2020) 17:71 Page 4 of 13 Results stratified into pre- and during-exercise intervention, a Energy intake, body weight and composition, and WAT significant interaction between time and diet was gene expression observed (Fig. 1b; F (1, 12)=9.42, p = 0.01) which ap- Over the 7-week study, cafeteria-fed rats ate more than peared to be due to increased cafeteria diet intake while twice the energy consumed by chow-fed groups (Fig. 1a; rats were exercising, although this comparison did not CSed: 18990 kJ/rat; CEx: 19380 kJ/rat; CafSed: 49234 kJ/ reach statistical significance. Significant interactions be- rat; CafEx: 53485 kJ/rat). When energy intake was tween time and diet were also identified for fat and Fig. 1 Short-term treadmill exercise reduces body weight gain and fat mass and alters WAT expression without affecting energy intake. a Energy intake over the study and b average weekly energy intake before and during treadmill exercise intervention. c Body weight of the study and d body weight gain during treadmill exercise intervention. e Fat mass (as a percentage of body weight) and f absolute lean mass; Data expressed as mean ± SEM; n = 4 for cage data, n = 11–12 for individual data; data were analyzed by two-way ANOVA followed by Tukey-adjusted post-hoc testing. ap < 0.05 relative to CSed, bp < 0.05 relative to CEx, cp < 0.05 relative to CafSed Leigh et al. Nutrition & Metabolism (2020) 17:71 Page 5 of 13 carbohydrate intakes (Supplementary Figure 1A; F (1, Cafeteria diet also increased naso-anal length, girth, 12)=5.92, p = 0.032 and Supplementary Figure 1C; F (1, plasma insulin and triglycerides, with no effect of exer- 12)=6.83, p = 0.023 respectively) where both macronutri- cise (Table 1). Plasma leptin levels were significantly in- ent intakes increased in the cafeteria-fed rats during ex- creased by diet (F (1, 43)=185.1, p < 0.001) and reduced ercise. Protein intake, while unaffected by time or by exercise (F (1, 43)=4.64, p = 0.037). Unfasted blood exercise, was elevated by cafeteria diet (Supplementary glucose did not differ between groups (Table 1). Figure 1B; F (3, 12)=24.06, p < 0.001). Sucrose intake in- In line with plasma leptin, retroperitoneal and epidydi- creased over time (Supplementary Figure 1D; F (1, 6)= mal fat pad weights were significantly greater in cafe- 51.90, p < 0.001) but was not affected by exercise. teria- than chow-fed rats (F (1, 44)=90.21, p < 0.001 and All rats gained body weight over time (Fig. 1c) and F (1, 44)=161.41, p < 0.001 for retroperitoneal and epidy- cafeteria-fed rats gained significantly more than chow- dimal fat pads respectively), and were significantly re- fed controls prior to (F (1, 44)=61.54, p < 0.001) and dur- duced by exercise overall (F (1, 44)=5.73, p = 0.021 and F ing (Fig. 1d; F (1, 44)=57.70, p < 0.001) exercise. Exercise (1, 44)=4.92, p = 0.032 for retroperitoneal and epidydimal significantly reduced weight gain overall (F (1, 44)=8.97, fat pads respectively) (See Table 1). p = 0.004). THSD post-hoc comparisons showed that compared to CSed and CEx, CafSed (p < 0.001 and p < Microbial species diversity and microbiome composition 0.001) and CafEx (p = 0.038 and p < 0.001) gained signifi- Microbial species diversity was assessed using Shannon’s cantly more weight over the exercise intervention. Of diversity index, microbial species richness and microbial note, CafSed rats gained significantly more weight than species evenness. Shannon’s diversity and evenness were CafEx rats over the exercise intervention (p = 0.043). significantly reduced by cafeteria diet overall (F (1, 44)= Body composition data following 4 weeks of treadmill 5.202, p = 0.027; Fig. 2a and f (1, 44)=8.278, p = 0.006; exercise showed that relative fat mass was significantly Fig. 2c respectively) and THSD post-hoc comparisons increased by cafeteria diet (Fig. 1e; F (1, 44)=154.58, p < showed that CafEx rats exhibited reduced evenness rela- 0.001; absolute fat mass presented in Table 1) and re- tive to CEx (p = 0.017) and reduced Shannon’s diversity duced by exercise (F (1, 44)=6.35, p = 0.015). THSD relative to CSed (p = 0.040). No significant differences post-hoc comparisons revealed that relative fat mass in were observed for bacterial species richness (Fig. 2b). both cafeteria-fed groups were significantly greater than Microbiome composition at the OTU level was signifi- both chow-fed groups. Lean mass however showed only cantly affected by diet (Pseudo-F (1, 32)=12.17, p = an overall diet effect (Fig. 1f; F (1, 44)=27.34, p < 0.001) 0.030) and cage (Pseudo-F (3, 32)=1.55, p = 0.006) but with significantly more lean mass in cafeteria-fed rats not by exercise when assessed using 4-way PERM than chow-fed rats. ANOVA (999 permutations) and confirmed with non- Table 1 Anthropometric measures at tissue collection and plasma measures Measure CSed CEx CafSed CafEx Main Effects (p-value) Diet Exercise Interaction a,b a,b Terminal Body Weight (g) 475.4 ± 14.9 455.6 ± 31.4 625.5 ± 26.2 583.3 ± 12.4 < 0.001 0.073 0.512 Nasoanal Length (cm) 24.5 ± 0.2 24.4 ± 0.2 25.0 ± 0.2 25.0 ± 0.1b < 0.001 0.914 0.450 a,b a,b Girth (cm) 19.1 ± 0.2 18.9 ± 0.2 22.3 ± 0.6 21.5 ± 0.4 < 0.001 0.186 0.505 Liver score 0.17 ± 0.09 0.17 ± 0.09 1.96 ± 0.17a,b 2.08 ± 0.22a,b < 0.001 a,b a,b Heart Weight (g) 1.39 ± 0.04 1.34 ± 0.03 1.69 ± 0.07 1.62 ± 0.03 < 0.001 0.170 0.884 Absolute fat mass (g; EchoMRI) 47.51 ± 3.63 38.78 ± 3.09 139.23 ± 16.27a,b 107.35 ± 6.81a,b < 0.001 0.031 0.212 a,b a,b RP Fat Pad Weight (% BW) 2.22 ± 0.16 1.87 ± 0.15 5.87 ± 0.43 4.73 ± 0.47 < 0.001 0.022 0.142 Epidydimal Fat Pad Weight (% BW) 2.35 ± 0.14 2.12 ± 0.17 6.01 ± 0.54a,b 4.91 ± 0.21a,b < 0.001 0.032 0.126 Blood Glucose (mmol/L) 9.0 ± 0.4 9.7 ± 0.9 10.4 ± 0.5 10.2 ± 0.5 0.548 0.225 0.111 Plasma Insulin (ng/mL) 0.53 ± 0.12 0.27 ± 0.07 2.09 ± 0.32a,b 1.68 ± 0.43a,b < 0.001 0.246 0.797 Plasma Leptin (ng/mL) 4.72 ± 0.55 3.81 ± 0.34 18.64 ± 1.43a,b 15.21 ± 1.21a,b < 0.001 0.037 0.220 a,b Plasma Triglycerides (mmol/L) 1.16 ± 0.12 0.86 ± 0.08 3.52 ± 0.32 3.27 ± 0.25a,b < 0.001 0.056 0.226 Liver Triglycerides (mg/g tissue) 3.77 ± 0.49 3.65 ± 0.44 23.32 ± 2.52a,b 20.58 ± 2.41a,b < 0.001 0.423 0.465 Blood and plasma measures performed unfasted. Data expressed as mean ± SEM; n = 10–12. Data were analyzed using two-way ANOVA, followed by post-hoc multiple comparisons with a Tukey HSD correction. Liver score was analysed using a Kruskal-Wallis test followed by non-parametric Bonferroni-Dunn post-hoc testing a p < 0.05 relative to CSed, bp < 0.05 relative to CEx Leigh et al. Nutrition & Metabolism (2020) 17:71 Page 6 of 13 Fig. 2 (See legend on next page.) Leigh et al. Nutrition & Metabolism (2020) 17:71 Page 7 of 13 (See figure on previous page.) Fig. 2 Impact of cafeteria diet and treadmill exercise intervention on fecal microbiota and inferred microbiome function at 7 weeks. a Shannon’s diversity, b microbial species richness and c evenness. Data expressed as mean ± SEM; n = 11–12; data were analyzed by two-way ANOVA followed by Tukey-adjusted post-hoc testing. d Non-metric multidimensional scaling (Bray-Curtis, 1000 permutations) showing similarity between fecal microbiota samples at 7 weeks. e Muribaculum_OTU72 identified by DESeq2 (adjusted p < 0.05) as differentially abundant with exercise in chow-fed rats. f Relative abundance of OTU72 at 7 weeks. Data expressed as box-and-whisker plots (min, IQR, max); n = 11–12; data were analyzed using Kruskal-Wallis test followed by non-parametric Dunn-Bonferroni post-hoc testing. g Amino acid metabolism, h overall energy metabolism, i D-glutamine and D-glutamate metabolism and j one carbon pool by folate predicted using PICRUSt from fecal microbiome data at 7 weeks. Data are expressed as box-and-whisker plots (min, IQR, max); n = 11–12; were analyzed by Kruskal-Wallis tests (FDR-adjusted overall p-value to account for multiple relevant pathways included in analysis) followed by non-parametric Bonferroni-Dunn post-hoc comparisons. Post-hoc symbols: ap < 0.05 relative to CSed, bp < 0.05 relative to CEx, cp < 0.05 relative to CafSed metric multidimensional scaling (Fig. 2d). Supplemen- Hypothalamic gene expression was analyzed to deter- tary Figure 2 shows groups differences in microbiome mine if 4 weeks of moderate exercise was sufficient to composition at the Phylum level. reverse diet-induced changes in expression of genes in- DESeq2 analyses were used to identify OTUs differen- volved in feeding (Fig. 3b), blood brain barrier integrity tially enriched with exercise exposure amongst the top and pro-inflammatory signaling (Fig. 3c). A significant 200 OTUs. While exercise was not associated with dif- interaction effect was observed for Npy gene expression ferentially expressed OTUs in cafeteria-fed rats, Muriba- in the hypothalamus (F (1, 41)=7.58, p = 0.009), and culum_OTU72 was significantly depleted in CEx rats THSD showed that CafSed rats exhibited downregulated relative to CSed (Fig. 2e, relative abundance in Fig. 2f; Npy relative to CSed (p < 0.001), CEx (p < 0.001) and this OTU was originally classified as Akkermansia when CafEx rats (p < 0.001). No significant differences were aligned with the RDP reference library). Muribaculum_ observed for Pomc, Agrp, Npy1r, Lepr or Insr. Crh gene OTU72 was putatively identified as an unknown bacter- expression was significantly increased in cafeteria-fed ium from the genus Muribaculum using SINA Aligner rats overall (F (1, 41)=14.89, p < 0.001), but was un- (97.6% alignment identity). affected by exercise (F (1, 41)=1.06, p = 0.309). Post-hoc THSD analysis revealed that CafEx rats exhibited signifi- cantly upregulated Crh relative to CSed (p = 0.001) and Predicted microbiome function CEx (p = 0.001). To determine whether the subtle microbiome compos- Both Cln5 and Glut1 were significantly increased in ition changes observed with exercise affected micro- cafeteria-fed rats overall (F (1, 41)=10.96, p = 0.002 and biome function, we inferred microbiome function using F (1, 41)=5.44, p = 0.025 respectively). While no group PICRUSt. Following an FDR correction, amino acid differences were apparent for Cln5 using THSD compar- metabolism (Fig. 2g; H (4)=16.3, p = 0.001), overall isons, Glut1 expression was significantly elevated in energy metabolism (Fig. 2h; H (4)=12.64, p = 0.006), D- CafSed rats relative to CSed controls (p = 0.045). No sig- glutamine and D-glutamate metabolism (Fig. 2i; H (4)= nificant differences were observed in Ocln, Tjp1 or any 14.58, p = 0.002) and one carbon pool by folate (Fig. 2j; of the pro-inflammatory genes assessed. H (4)=11.44, p = 0.010) exhibited overall group differ- ences. Amino acid metabolism was significantly elevated Associations between variables of interest and in CafEx relative to CSed (p < 0.001) and CafSed (p = microbiome 0.023) rats while the 3 other processes were significantly When variables were assessed for their unique contribu- elevated in both exercised groups relative to CSed. tion to the variance in overall microbiome composition, several adiposity measures, as well as retroperitoneal WAT and hypothalamic gene expression WAT Il6 gene expression and hypothalamic Crh and Examination of WAT inflammatory signaling and Npy expression, were identified as significant predictors browning genes (Fig. 3a) revealed a significant inter- of global microbiome composition (Supplementary action effect for Ucp1 (F (1, 40)=4.41, p = 0.042) while Table 2). When the contribution of variables of interest THSD post-hoc comparisons showed that CafEx rats to overall microbiome composition was assessed while exhibited elevated Ucp1 expression relative to CafSed controlling for diet and treadmill exercise, both liver (p = 0.024). Lep expression was significantly elevated triglyceride concentration (R2 = 0.031, p = 0.008) and by cafeteria diet consumption (F (1, 41)=4.98, p = retroperitoneal WAT Il6 gene expression (R2 = 0.026, 0.031), while Lepr expression was increased with exer- p = 0.027) were significant predictors of microbiome cise overall (F (1, 41)=4.75, p = 0.035). No significant composition at the OTU level (Table 2, complete differences were observed in pro-inflammatory model predicts 34.7% of the variance in microbiome markers or Adipoq gene expression. composition). Leigh et al. Nutrition & Metabolism (2020) 17:71 Page 8 of 13 Fig. 3 Gene expression in retroperitoneal WAT, hypothalamus and associations with gut microbiome changes. a Adipokine, metabolic and inflammatory gene expression in retroperitoneal WAT. b Feeding- and stress-related gene expression in the hypothalamus. c Blood-brain barrier and pro-inflammatory gene expression in the hypothalamus. Adipoq: adiponectin, Agrp: Agouti-related protein, Cln5: Claudin-5, Crh: Corticotrophin releasing hormone, Glut1: Glucose transporter 1, Il6: interleukin 6, Il10: interleukin-10, Il1b: interleukin 1 beta, Insr: Insulin receptor, Lep: Leptin, Lepr: Leptin receptor, Npy: Neuropeptide y, Npy1r: Neuropeptide y receptor 1, Ocln: Occludin, Pomc: Pro-opiomelanocortin, Tjp1: Tight junction protein 1, Tnf: Tumour necrosis factor, Ucp1: uncoupling protein 1; Data expressed as mean ± SEM; n = 11–12; data were analyzed by two-way ANOVA followed by Tukey-adjusted post-hoc testing. ap < 0.05 relative to CSed, bp < 0.05 relative to CEx, cp < 0.05 relative to CafSed Porphyromonadaceae unclassified_OTU106 was in- Fig. 4d) and was positively associated with Porphyromo- creased with cafeteria diet (Fig. 4a; H (4)=11.42, p = nadaceae unclassified_OTU106 (Fig. 4e), following a 0.010) and significantly associated with WAT Il6 gene similar, although non-significant, trend to that observed expression (Fig. 4b). Interestingly, this OTU was also with Il6 gene expression. Like Il6 gene expression, WAT negatively correlated with hypothalamic Npy gene IL-6 content was a significant independent predictor of expression (Fig. 4c). IL-6 protein in WAT exhibited a overall microbiome composition (Supplementary Table significant interaction effect (F (1, 41)=5.310, p = 0.026; 2) but was not a significant predictor in the final model, Leigh et al. Nutrition & Metabolism (2020) 17:71 Page 9 of 13 Table 2 The shared contributions of diet, exercise and variables of biological relevance on the variance observed in microbiome composition using distance-based linear modelling Variable SS Pseudo-F P-Value R2 Cumulative R2 Diet 18,456 14.502 0.001 0.266 0.266 Exercise 1686.7 1.337 0.073 0.024 0.290 Liver triglyceride content 2157.7 1.742 0.008 0.031 0.321 WAT Il6 expression 1822 1.490 0.027 0.026 0.347 Sequential multiple regression (captured by the Bray-Curtis similarity matrix at the OTU level, max 1000 permutations) involves interrogating the conditional contribution of each variable in order of entry into the model (to determine whether variables contribute significantly to the variance explained in the presence of other variables); here, diet and exercise conditions were added before any metabolic predictors were considered and the final model containing only statistically significant covariates is shown. Metabolic predictors included in the sequential regression were selected based on their predictive value, while trying to eliminate variables with high covariance; N = 42–46 after controlling for diet and exercise. SINA Aligner induced effects on microbiome composition here may be putatively identified Porphyromonadaceae unclassified_ more resistant to the effects of exercise than previously OTU106 as a strain of Bacteroides eggerthii, a Gram- reported. Additionally, a number of rodent studies re- negative bacterium known to hydrolyze carbohydrates port no differences in overall microbiome composition including simple sugars [32]. with exercise [17, 19], and there is evidence that this ef- fect may be moderated by age [39] which may have con- Discussion tributed to the inconsistent findings in the literature. We found that 4 weeks of moderate treadmill exercise Predicted microbial functions associated with metabol- reduced fat mass and plasma leptin concentrations and ism, specifically overall energy metabolism, amino acid altered WAT expression of some adipokine- and metabolism, one carbon pool by folate and D-glutamine metabolism-associated genes. Overall microbiome com- and D-glutamate metabolism, were increased in exer- position and microbial species diversity was changed by cised rats. This is in line with metagenomic results cafeteria diet but not by exercise. However, predicted where fecal microbiome from male elite athletes exhib- microbial functions associated with metabolism were in- ited increased amino acid biosynthesis and overall en- creased by exercise. Cafeteria diet-induced changes in ergy metabolism relative to sedentary, normal-weight hypothalamic Npy and Glut1 gene expression were controls [40]. While PICRUSt analysis produces pre- returned to control levels by exercise. dicted functional data, unlike metagenomic analysis, this Exercise induced modest changes in gut microbiome is an interesting finding that warrants follow-up to de- composition that were statistically significant in chow- termine if and how exercise shifts the metabolic profile fed rats only and the relative abundance of OTU72 was of the gut microbiome and whether any such shift is af- significantly reduced in chow-fed rats that exercised fected by exercise intensity and duration. Furthermore, (CEx) rats relative to sedentary controls (CSed). While confirming these results across a range of diets would be CafEx rats exhibited reduced microbial species diversity, useful to determine whether the shift in microbial func- this reduction appeared dependent on cafeteria diet tion with exercise is modulated by the macro- and exposure rather than exercise. To date work examining micronutrients available. the effects of exercise on microbial species diversity has Here, a moderate exercise intervention reduced fat produced inconsistent results [15]: some rodent studies mass and plasma leptin concentrations, and increased have shown that exercise is associated with reductions in WAT Lepr gene expression in both exercised groups fecal microbial species richness [16] while others have and Ucp1 gene expression in exercised, cafeteria-fed reported no such effect [33]. (CafEx) rats uniquely. Increased Ucp1 in WAT depots is After 4 weeks of treadmill exercise we did not observe a marker of adipocyte beiging [41], known to be pro- changes in overall microbiome composition. Our data moted by exercise [42], and is most likely related to are in line with findings in humans showing that exercise-induced fat loss. While there were no significant changes in microbiome composition are dependent on effects of exercise on WAT pro-inflammatory gene ex- obesity status, such that more severe obesity is associ- pression, after controlling for diet and exercise WAT Il6 ated with smaller effects of exercise [34]. Since the expression was significantly associated with global cafeteria diet used here [20] tends to produce a more se- microbiome composition. vere metabolic phenotype than purified high-fat diets WAT is one of the major sources of IL-6 in obese [35–38], which to our knowledge have been used in all humans [43] and mice [44, 45], which is a key compo- studies investigating the interrelationship between exer- nent of the low-grade systemic inflammation observed cise and diet on microbiome composition, the diet- in overweight and obesity [46] and is associated with Leigh et al. Nutrition & Metabolism (2020) 17:71 Page 10 of 13 Fig. 4 Relationship between OTU106, and hypothalamic Npy and WAT IL-6 expression (a) Relative abundance of OTU106; data expressed as box- and-whisker plots (min, IQR, max); n = 11–12; data were analyzed using Kruskal-Wallis test followed by non-parametric Dunn-Bonferroni post-hoc testing. Scatterplots for OTU106 abundance and (b) Il6 gene expression in WAT, and (c) Npy gene expression in the hypothalamus, showing overall lines of best fit; n = 46. d WAT IL-6 protein content; data expressed as mean ± SEM; n = 10–12; data were analyzed by two-way ANOVA followed by Tukey-adjusted post-hoc testing. e Scatterplot for OTU106 abundance and IL-6 protein content in WAT, showing overall lines of best fit; n = 45. ap < 0.05 relative to CSed, bp < 0.05 relative to CEx, cp < 0.05 relative to CafSed insulin resistance [47]. WAT Il6 expression was strongly associations between WAT Il6 expression, global micro- associated with the relative abundance of a strain of biome composition and OTU106 abundance are there- Bacteroides eggerthii (OTU106), a Gram-negative sugar- fore likely to be due to the effects of cafeteria diet on scavenging bacterium [32] which is enriched in obese both adipose inflammatory processes and the gut micro- children relative to normal-weight controls [48]. The biome. However, probiotic treatment with a strain of Leigh et al. Nutrition & Metabolism (2020) 17:71 Page 11 of 13 Bifidobacterium in mice fed a high-fat diet reduced Conclusions WAT macrophage infiltration and plasma IL-6 concen- In summary, short-term (4 weeks) treadmill exercise in- tration [49], indicating that changes to the gut micro- duced subtle microbiome composition changes in chow- biome may contribute to WAT inflammatory signaling fed rats but did not overcome the microbiome changes and IL-6 production. Further studies determining induced by cafeteria diet. Despite this, predicted micro- whether specific bacterial species can modulate WAT biome function was altered by exercise in both chow- IL-6 production are warranted, as interventions that and cafeteria-fed groups. Exercise reduced adiposity and could reduce WAT IL-6 expression in obesity may pro- altered both WAT and hypothalamic gene expression. vide an avenue for preventing insulin resistance and type WAT Il6 expression was significantly associated with 2 diabetes. microbiome composition and negatively correlated with In contrast to the overall effect of exercise on WAT the abundance of a strain of Bacteroides eggerthii, bac- gene expression, hypothalamic genes disrupted by cafe- teria known to scavenge sugars. Future work identifying teria diet were typically normalized with exercise, with the circumstances where exercise exerts effects on the no differences observed in chow-fed rats. Npy was microbiome, and the potential causal role of these downregulated in CafSed rats, as shown previously [50], changes for reducing diet- and obesity-associated co- but normalized to control levels with exercise, which is morbidities, is warranted. in line with other rodent work showing increased hypo- thalamic NPY mRNA and protein in response to both Supplementary information acute and chronic exercise [51–54]. Hypothalamic Glut1 Supplementary information accompanies this paper at https://doi.org/10. gene expression was increased in CafSed rats and re- 1186/s12986-020-00492-6. duced to control levels with exercise. This is in contrast Additional file 1: Supplementary Table 1. Taq assay probe to studies showing that acute exercise increased GLUT1 information. Supplementary Figure 1. Weekly macronutrient intake protein expression across the rat brain [55] and pro- and sucrose intake over the study. Supplementary Figure 2. Average longed exercise increases whole brain resting glucose composition of phyla between experimental groups. Supplementary Table 2. Distance-based linear modelling to determine contributions of uptake in people [56]. Further work investigating acute metabolic and gene expression measures to the variance observed in and chronic exercise-induced changes in Glut1 expres- microbiota composition at the OTU level. sion in the hypothalamus and other brain regions is required. Abbreviations Cafeteria diet-induced Crh upregulation in the hypo- Adipoq: Adiponectin; Agrp: Agouti-related peptide; B2m: Beta-2-microglobin; thalamus was not normalized by exercise. Crh is CEx: Chow Exercise; Cln5: Claudin 5; Crh: Corticotrophin-releasing hormone; CSed: Chow Sedentary; CafEx: Cafeteria Exercise; CafSed: Cafeteria Sedentary; transcribed to corticotrophin-releasing hormone (CRH), dbLM: Distance-based linear modelling; FDR: False discovery rate; which activates the hypothalamic-pituitary-adrenal Gapdh: Glyceraldehyde 3-phosphate dehydrogenase; Glut1: Glucose (HPA) axis, key in physiological and behavioral re- transporter 1; Hprt1: Hypoxanthine phosephoribosyltransferase 1; Il6: Interleukin-6; Insr: Insulin receptor; Lep: Leptin; Lepr: Leptin receptor; sponses to stress. Involuntary exercise is a known stres- nMDS: Non-metric multidimensional scaling; Npy: Neuropeptide Y; sor [57], and forced exercise increased activity in CRH Npy1r: Neuropeptide Y receptor 1; Ocln: Occludin; OTU: Operational neurons relative to both voluntary exercise and seden- taxonomic unit; PERMANOVA: Permutational multivariate ANOVA; PICRUSt: Phylogenetic investigation of communities by reconstruction of tary control groups in rats [58]. unobserved states; Pomc: Pro-opiomelancortin; Tjp1: Tight junction protein 1; Exercise can impact the gut microbiome via several THSD: Tukey honest significant difference; WAT: White adipose tissue; pathways, and there are multiple potential mechanisms Ucp1: Uncoupling protein 1 by which these gut microbiome changes could subse- Acknowledgements quently confer health benefits. Exercise may shape The authors thank Robyn Lawler at the Biological Resources Centre, UNSW microbiome composition and function by increasing Sydney, for assistance with animal husbandry, Dr. Sabiha Chowdhury, Saroj host metabolic requirements and shifting metabolite Khatiwada and Josephine Yu for assistance with dissection. This research includes data processed by the computational cluster Katana supported by availability. A recent study showed that marathon run- Research Technology Services at UNSW Sydney. ners exhibit increased abundance of Veillonella atypica, which rely on lactate as a source of carbon, and inocula- Authors’ contributions tion with this species increased running time in mice Conceived and designed experiments: SJL, RFW, MJM. Performed the [59]. Alternatively, exercise is known to activate the experiments: SJL, RME. Behavioral data, PCR: SJL. Microbiome data: SJL, NOK. Interpretation of data and writing: SJL, NOK, RFW, MJM. All authors approved HPA axis [60]. The HPA axis appears to exert effects on the manuscript. the gut microbiome, and may be impacted by micro- biome modification [61]. Therefore, the effects of exer- Funding cise on stress behaviors and physiology may be in part The work was supported by an NHMRC project grant (#1126929) to MJM and RFW as well as a Brain Sciences UNSW seed grant to SJL, MJM and RFW. due to exercise-induced changes to the gut microbiome, SJL was supported by a Research Training Program scholarship. RME was or vice versa. supported by the project grant PSI2016–77234-R from Ministerio de Leigh et al. Nutrition & Metabolism (2020) 17:71 Page 12 of 13 Economía y Competitividad and by the grant PRX15/00455 from Ministerio 16. Allen JM, Miller MEB, Pence BD, Whitlock K, Nehra V, Gaskins HR, et al. de Educación, Cultura y Deporte. Voluntary and forced exercise differentially alters the gut microbiome in C57BL/6J mice. J Appl Physiol. 2015;118(8):1059–66. Availability of data and materials 17. Lamoureux EV, Grandy SA, Langille MGI. Moderate Exercise Has Limited but The study metadata and sequence data are available in the European Distinguishable Effects on the Mouse Microbiome. mSystems. 2017;2(4): Nucleotide Archive under accession number PRJEB36541. The study is e00006–17. currently set on private and will be released upon acceptance. All other data 18. Kang SS, Jeraldo PR, Kurti A, Miller ME, Cook MD, Whitlock K, et al. Diet and will be made available from the corresponding author on reasonable exercise orthogonally alter the gut microbiome and reveal independent request. associations with anxiety and cognition. Mol Neurodegener. 2014;9:36. 19. Ribeiro FM, Ribeiro CFA, G ACM, Castro AP, Almeida JA, Franco OL, et al. Ethics approval and consent to participate Limited Effects of Low-to-Moderate Aerobic Exercise on the Gut Microbiota This protocol was approved by the Animal Care and Ethics Committee of of Mice Subjected to a High-Fat Diet. Nutrients. 2019;11(1):149. 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