Gut microbiota Gut: first published as 10.1136/gutjnl-2019-319114 on 19 November 2019. Downloaded from http://gut.bmj.com/ on November 20, 2019 at Karolinska BIBSAM Consortia. Protected by copyright. Original article Myristoleic acid produced by enterococci reduces obesity through brown adipose tissue activation Lin-Hu Quan,1 Chuanhai Zhang,2 Meng Dong,2,3 Jun Jiang,1 Hongde Xu,4 Chunlong Yan,1 Xiaomeng Liu,5 Huiqiao Zhou,2,3 Hanlin Zhang,2,3 Li Chen,2,3 Fei-Liang Zhong,1 Zhao-B o Luo,6 Sin-M an Lam,7 Guanghou Shui,7 Donghao Li,1 Wanzhu Jin 2,3 ►► Additional material is ABSTRACT Significance of this study published online only. To view Objective Dietary fibre has beneficial effects on please visit the journal online energy metabolism, and the majority of studies have What is already known on this subject? (http://d x.doi.o rg/10.1 136/ focused on short-chain fatty acids produced by gut ►► Ginseng extract (GE) has an anti-o besity effect. gutjnl-2 019-319114). microbiota. Ginseng has been reported to aid in body ►► The activation of thermogenesis in brown For numbered affiliations see weight management, however, its mechanism of end of article. action is not yet clear. In this study, we focused on the adipose tissue (BAT) and beige fat is beneficial potential modulating effect of ginseng on gut microbiota, for weight management. Correspondence to aiming to identify specific strains and their metabolites, ►► Modulation of gut microbiota using prebiotics Professor Wanzhu Jin, Key especially long-chain fatty acids (LCFA), which mediate and probiotics may improve host metabolism Laboratory of Animal Ecology the anti-obesity effects of ginseng. and reduce obesity. and Conservation Biology, Design Db/db mice were gavaged with ginseng What are the new findings? Institute of Zoology, Chinese extract (GE) and the effects of GE on gut microbiota ►► GE exhibited an anti-obesity effect by activating Academy of Sciences, Beijing, were evaluated using 16S rDNA-b ased high throughput BAT. Beijing, China; jinw@ioz.ac.cn sequencing. To confirm the candidate fatty acids, ►► GE specifically increased the abundance of E. L-H Q, CZ, MD, JJ, HX and CY untargeted metabolomics analyses of the serum and faecalis in the gut and E. faecalis administration contributed equally. medium samples were performed. reduced body weight through BAT activation. Received 18 May 2019 Results We demonstrated that GE can induce ►► E. faecalis facilitated its beneficial effects on Revised 21 October 2019 Enterococcus faecalis, which can produce an unsaturated metabolism through its metabolites; the most Accepted 6 November 2019 LCFA, myristoleic acid (MA). Our results indicate that important one being myristoleic acid (MA), a E. faecalis and its metabolite MA can reduce adiposity long-chain fatty acid. by brown adipose tissue (BAT) activation and beige ►► Acyl-CoA thioesterase is at least partially fat formation. In addition, the gene of E. faecalis responsible for the biosynthesis of MA in E. encoding Acyl-CoA thioesterases (ACOTs) exhibited the faecalis. biosynthetic potential to synthesise MA, as knockdown How might it impact on clinical practice in the (KD) of the ACOT gene by CRISPR-d Cas9 significantly foreseeable future? reduced MA production. Furthermore, exogenous ►► E. faecalis, as a novel anti-obesity probiotic, treatment with KD E. faecalis could not reproduce the can be exploited to manage excess weight and beneficial effects of wild type E. faecalis, which work by obesity. augmenting the circulating MA levels. ►► MA is a new postbiotic from E. faecalis with Conclusions Our results demonstrated that the gut beneficial effects on obesity and its related microbiota-LCFA-BAT axis plays an important role in host diseases. metabolism, which may provide a strategic advantage for the next generation of anti-obesity drug development. © Author(s) (or their Introduction that brown adipose tissue (BAT) facilitates weight employer(s)) 2019. No Obesity has become a global epidemic. It leads to an control, health, and provides an anti-obesity effect.4 commercial re-u se. See rights increased risk of various diseases including insulin Therefore, increasing BAT activity could be a novel and permissions. Published resistance, type 2 diabetes, fatty liver disease, cardio- and effective therapeutic approach for preventing by BMJ. vascular disease and certain types of cancers.1 2 and curing obesity and its related diseases.5–7 To cite: Quan L-H, Zhang C, Obesity develops due to higher energy intake than Dong M, et al. Gut Epub energy expenditure, which results in excess energy Intestinal flora is an important environmental ahead of print: [please storage in white adipose tissue (WAT) in the form factor for energy acquisition and storage in obesity, include Day Month Year]. of triglycerides.3 The current anti-o besity strategies and thus, it has an impact on the host.8 9 Interestingly, doi:10.1136/ are mainly aimed at restricting energy uptake and there are reports demonstrating that acclimation to gutjnl-2019-319114 absorption. However, obesity is far from being dealt cold enhances host metabolism by modulating gut with satisfactorily. Previous studies have shown microbiota.10 Therefore, strategies that modulate the gut microbiota have been proposed to prevent and treat obesity. Previous studies have shown that modulation of gut microbiota using prebiotics and Quan L-H, et al. Gut 2019;0:1–9. doi:10.1136/gutjnl-2019-319114 1
Myristoleic acid produced by enterococci reduces obesity through brown adipose tissue activation
Gut microbiota Beijing, China). GE (10 mg/kg), bacterial strains (1010 CFU/day, Gut: first published as 10.1136/gutjnl-2019-319114 on 19 November 2019. Downloaded from http://gut.bmj.com/ on November 20, 2019 at Karolinska BIBSAM Consortia. Protected by Aoke Biology Research, Beijing, China), or fatty acids (5 mg/kg, copyright. probiotics may improve host metabolism and reduce obesity ANPEL Laboratory Technologies, Shanghai, China) were admin- and associated metabolic diseases. Examples include the effect istered by oral gavage during the indicated period starting from of polysaccharides and dietary fibres in reducing body weight the age of 4–6 weeks. For in vivo antibiotic treatment, db/db and alleviating type 2 diabetes by inducing specific probiotics mice were treated with combined antibiotics (ABX) (containing and their derived metabolites including short-chain fatty acid 100 µg/mL neomycin (Sigma), 50 µg/mL streptomycin (Sigma), (SCFA) and probiotic strains such as Lactobacillus and Bifidobac- 100 U/mL penicillin (Sigma), 50 µg/mL vancomycin (Sigma), terium, which can attenuate obesity comorbidities.11 12 In addi- 100 µg/mL metronidazole (Sigma), 1 mg/mL bacitracin (Sigma), tion, it has been reported that long-chain fatty acids (LCFAs), 125 ug/mL ciprofloxacin (Sigma), 170 ug/mL gentamycin (Sigma) which are defined as saturated and/or unsaturated fatty acids and 10 ug/mL chloramphenicol (Lablead, Beijing, China)) in with 14–20 carbons, also play important roles in regulating sterile water for 5 days. After ABX treatment, the ACOT WT the energy metabolism.13 There are reports that eicosapentae- and KD E. faecalis strains were fed by oral gavage for 1 week noic acid induces thermogenesis in brown adipocytes through (1010 CFU/day). FFAR4-dependent upregulation of miR-30b and miR-378,14 and myristic acid improves hyperglycaemia in a mouse model of All the mice were housed in groups of 4–5 animals per cage type 2 diabetes.15 Besides, LCFAs, which are produced in brown in a pathogen-free facility with a 12 hours: 12 hours light: dark adipocytes by the lipolysis, can activate uncoupling protein 1 cycle with ad libitum access to food and water. In all the exper- (UCP1) in thermogenic fat.15 Importantly, a recent study indi- iments, the mice were fasted overnight before euthanasia using cated that LCFAs are essential for UCP1 uncoupling.16 mild ether anaesthesia, and sacrificed. All the animal studies were approbated by the Institutional Animal Care and Use Committee Traditional Chinese Medicine (TCM), often administered of Institute of Zoology (Chinese Academy of Sciences). orally, has been widely used in the treatment of various diseases and dates back to thousands of years. TCM (such as Ganoderma Analysis of gut microbiota lucidum) can modulate the gut microbiota composition by The gut microbiota was analysed as previously described.22 increasing probiotics and reducing pathogens, thus preventing Briefly, genomic DNA (0.25 g) was isolated from bacterial colo- the development and progression of obesity and other diseases.17 nies with the PowerSoil DNA Isolation Kit (MO BIO Labora- However, its mechanism of action is not yet clear. Panax ginseng is tories, USA). After amplification and purification of the V3-V 4 widely used as a traditional medicine or functional food.18 19 The region of bacterial 16 s rRNA genes, the abundance and diver- ginsenosides are the major active components of Panax ginseng, sity of intestinal flora in mice were determined using Illumina responsible for various biological functions including effects HiSeq sequencing (Novogene, Beijing, China). The raw reads against obesity and obesity-related diseases,20 and are metabo- have been submitted to the BIGD Genome Sequence Archive lised to rare saponins through deglycosylation reactions by the database (Accession number CRA001467). Sequencing libraries gut microbiota before being absorbed into the blood (eg, ginse- were assessed utilising Agilent 2100 bioanalyzer (Agilent Tech- noside CK).21 22 In addition, a study has shown that long-term nologies, USA) and the qualified libraries were amplified on cBot administration of ginseng extracts (GEs) could change the gut to obtain the gene clusters on a flow-c ell. microbiota composition.23 Therefore, further studies are needed to better understand the complex interactions between ginseng According to the unique barcodes, paired-e nd reads were allo- and gut microbiota and secondary metabolites and obesity. cated to the samples. The overlapped regions between paired-end reads were combined using FLASH v1.2.7. According to the In the present study, we revealed that GE significantly increased QIIME (V1.7.0) quality control process, high-quality clean tags energy expenditure and reduced adiposity via BAT activation. were obtained by qualitatively filtering the raw reads under Furthermore, GE induced the intestinal bacterium E. faecalis, specific filtering conditions. Based on the UCHIME algorithm, which can produce myristoleic acid (MA), which reduces obesity the chimaera sequences were detected by comparing tags with the through increasing BAT activity and beige fat formation. Addi- Gold database and then removed. The QIIME software package tionally, we found that the Acyl-C oA thioesterase (ACOT) gene was used to conduct the bioinformatic analyses of the sequences. of E. faecalis has a potential for MA biosynthesis. Thus, our data Sequences sharing at least 97% comparability were attributed indicated that E. faecalis and its metabolite, MA, might be used to the same operational taxonomic units (OTUs). QIME was for the prevention and treatment of obesity and its complications. used to carry out alpha, beta diversity and Principal Coordinates Analysis depending on the unweighted unifrac distances. Methods Mice Establishment of ACOT gene knockdown strain of E. faecalis Four-week-old male C57BLKS/J-Leprdb/Leprdb (db/db) mice The ACOT gene of E. faecalis was knocked down using the were purchased from the Model Animal Research Center of CRISPR Interference System according to the method established Nanjing University. Four-week-old male C57BL/6J mice were by Qi.25 The dCas9 gene was cloned from the plasmid pHR- obtained from Vital River Laboratory Animal Technology SFFV-dCas9-BFP-K RAB (46911, Addgene). The sgRNA scaffold (Beijing, China). UCP1 luciferase transgenic mice (Thermo- with J23119 promotor was synthesised (Genscript, Nanjing, Mouse) were purchased from Jackson Labs (Bar Harbor, Maine, China). The genes were amplified and cloned into the vector USA) and backcrossed with C57BL/6J mice. The UCP1 KO mice SlpA −8148 (a gift from Dr Yanling Hao) containing a consti- (genetic background C57Bl/6J) were originally established by tutive promoter SlpA and a chloramphenicol-selectable marker. L Kozak (Pennington Medical Research Centre, Baton Rouge, The ACOT gene knockdown (KD) strain was created by electro- Louisiana, USA). WT and UCP1 KO mice were obtained from transformation with dCas9-expressing and ACOT-targeting heterozygous breeding pairs in our SPF laboratory animal-h ouse sgRNA (sequence: ATCCCTTTGGTGCACTATT) vector, while (Institute of Zoology, Chinese Academy of Sciences, Beijing, the WT strain was created by electro-transformation with a China) and the genotype was evaluated as described previ- dCas9-expressing and sgRNA scaffold vector. Chloramphenicol ously.24 Each group of mice was fed a chow diet or a high-f at diet (HFD; 60% kcal fat as indicated, Beijing HuaFuKang Bioscience, Quan L-H , et al. Gut 2019;0:1–9. doi:10.1136/gutjnl-2019-319114 2
Gut microbiota Gut: first published as 10.1136/gutjnl-2019-319114 on 19 November 2019. Downloaded from http://gut.bmj.com/ on November 20, 2019 at Karolinska BIBSAM Consortia. Protected by copyright. Figure 1 Ginseng extract (GE) increases whole-body energy metabolism and BAT activity in db/db mice. Db/db mice were treated with GE (10 mg/ kg) for 8 weeks by daily oral gavage (n=7–8). (A–C) GE treatment significantly decreased body weight gain as well as the EP organ weight of db/ db mice. (D–F) Energy expenditure and core body temperature were significantly increased after GE treatment in db/db mice. (G–I) GE treatment significantly increased UCP1 expression and mitochondrial oxphos protein expression in BAT and Sub of db/db mice. For body weight curves, statistical analysis was performed using a repeated measure two-way ANOVA, for energy expenditure, ANCOVA were employed and the rest of the statistics was performed with Student’s t-test. Data are means±SEM *p<0.05; **p<0.01; NS, not statistically significant. ATP, adenosine triphosphate; BAT, brown adipose tissue; EP, epididymal fat; SDHB, succinate dehydrogenase complex,subunit B; UCP, uncoupling protein; UQCRC, ubiquinol cytochrome c reductase core protein. (10 µg/mL) was used to select the positive clones. After culturing compared with those receiving the vehicle treatment (figure 1A). in MRS medium at 37°C overnight, the total RNA was isolated The body composition was analysed using CT. Consistent with using a bacteria RNA Extraction Kit (R403-1, Vazyme, Nanjing, body weight change, the percentage of whole-body fat decreased China) and cDNA was synthesised with HiScript III first Strand by 11% compared with that of the control mice (figure 1B). cDNA Synthesis Kit (R312-01, Vazyme, Nanjing, China). The Moreover, the organ weight of epididymal fat was significantly ACOT gene expression level was detected as described above, decreased after GE treatment (figure 1C), whereas the organ while 16S rDNA was chosen as an internal reference. weight of BAT, subcutaneous fat (Sub), and liver did not show significant changes (figure 1C). These results indicated that GE Statistics treatment significantly reduced body weight and adiposity in db/ Data are expressed as means±SE. Statistics were performed using db mice. Adiposity is often accompanied with an alteration of a repeated measure two-w ay analysis of variance (ANOVA), the energy balance. Consequently, we examined whether GE the one-way ANOVA test, analysis of covariance (ANCOVA) treatment could affect energy metabolism via indirect calorim- or Student’s t-test. Statistical significance was set at p<0.05. etry (using a TSE labmaster system). Interestingly, GE treatment *p<0.05; **p<0.01; ***p<0.0001. significantly increased energy expenditure compared with the control mice (figure 1D), while there was no significant differ- Results ence in the food intake or energy intake (online supplementary GE decreases adiposity and enhances energy metabolism by figure 1A,D), physical activity (online supplementary figure BAT activation 1B), or respiratory exchange ratio (RER) (online supplemen- In this study, we assessed the possibility of the anti-obesity tary figure 1C), absorbed energy (online supplementary figure effects of GE mediated by gut microbiota and BAT. GE (10 mg/ 1E) and no absorbed energy (online supplementary figure 1F) kg) was administered to db/db mice by daily oral gavage. After between the two groups. The results of energy balance strongly 8 weeks, their body weight gain were significantly decreased indicated that the anti-obesity role of GE is due to increased Quan L-H , et al. Gut 2019;0:1–9. doi:10.1136/gutjnl-2019-319114 3
Gut microbiota we speculated on whether gut microbiota could play key anti- Gut: first published as 10.1136/gutjnl-2019-319114 on 19 November 2019. Downloaded from http://gut.bmj.com/ on November 20, 2019 at Karolinska BIBSAM Consortia. Protected by obesity roles in GE-treated mice using this mode of action. copyright. energy expenditure rather than reduced food intake or increased Towards this end, faecal samples were examined for bacterial physical activity. Furthermore, GE treatment greatly increased community diversity via 16S rRNA gene amplicon sequencing. core body temperature when animals were exposed to a cold The α-diversity values for the bacterial community analysis with environment (4°C, 4 hours) (figure 1E,F). Importantly, GE ACE, Chao 1, Shannon index and Simpson index in the GE treatment improved glucose homoeostasis (online supplemen- treated mice were significantly lower than those in the control tary figure 1G) and insulin sensitivity(online supplementary mice (online supplementary table 1). The overall structure of figure 1H）, and reduced hepatic steatosis (online supplemen- the gut microbiota that was investigated by principal compo- tary figure 1I–K). These results indicated that GE treatment nent analysis showed that the two groups were clearly differen- improved the energy homoeostasis in obese mice. This can be tiated (figure 2A). The phylum-level proportional abundances, partially explained by the improved BAT thermogenic activity especially within Firmicutes and Bacteroidetes, did not show (figure 1G) and the increased expression of UCP1, which gener- significant differences after GE treatment (online supplemen- ates heat through the uncoupling process and OXPHOS expres- tary figure 2A). This might be due to the limitations of the sion in BAT and Sub of the GE treated db/db mice (figure 1H,I). chosen technology; the 16S rRNA gene amplicon sequencing BAT is a thermogenic organ, essential in maintaining core body could not cover 100% of the gut microbiota. Beyond this, there temperature and its thermogenic activity is inhibited at thermo- were 35 OTUs that were significantly altered by GE in db/db neutrality.26 However, GE treatment did not affect body weight mice (online supplementary figure 2B). Interestingly, we found gain at thermoneutrality (online supplementary figure 1L). This that E. faecalis was considerably enriched at the family-g enus- result proved that the anti-o besity role of GE certainly depends species levels (figure 2B–D). Thus, our results indicated that the on the BAT activity. Taken together, these results indicated that composition of gut microbiota changed significantly in response GE increased whole body energy metabolism by BAT activation to GE treatment and that E. faecalis was substantially enriched without altering the energy intake or physical activity. (figure 2E). GE treatment enriches E. faecalis It was previously demonstrated that ginsenosides can be degly- cosylated by the actions of the gut microbiota.27 Therefore, Figure 2 Ginseng extract (GE) alters microbiota composition in db/db mice. Db/db mice were treated with GE (10 mg/kg) for 8 weeks by daily oral gavage (n=8). (A) Plots shown were generated using the weighted version of the UniFrac-b ased principal component analysis. (B–D) Comparison proportion of family, genus and species levels of E. faecalis in faeces detected by pyrosequencing analysis. (E) Discriminative taxa determined by LEfSe between two groups (log10 LDA >3.5). Statistical analysis was performed using Student’s t-test between db/db and db/db+GE groups. Data are means±SEM *p<0.05. LDA, linear discriminant analysis, 4 Quan L-H, et al. Gut 2019;0:1–9. doi:10.1136/gutjnl-2019-319114
Gut microbiota Gut: first published as 10.1136/gutjnl-2019-319114 on 19 November 2019. Downloaded from http://gut.bmj.com/ on November 20, 2019 at Karolinska BIBSAM Consortia. Protected by copyright. Figure 3 E. faecalis (EF) increases whole-body energy metabolism and BAT activity in HFD-fed mice. HFD-fed mice were treated daily with EF (1×1010 CFU) by oral gavage for 8 weeks (n=7–9). (A–B) EF treatment significantly decreased body weight gain as well as the fat mass of HFD-fed mice. (C–E) Core body temperature and energy expenditure were significantly increased after EF treatment in HFD-fed mice. (F–H) EF treatment significantly increased UCP1 expression and mitochondrial oxphos protein expression in bat and sub of HFD-fed mice. For body weight curves, statistical analysis was performed using a repeated measure two-w ay ANOVA, for energy expenditure, ANCOVA were employed and the rest of the statistics was performed with Student’s t-test. Data are means±SEM.*p<0.05; **p<0.01; ***p<0.0001; NS, not statistically significant. ATP, adenosine triphosphate; BAT, brown adipose tissue; EP, epididymal fat; HFD, high-fat diet; HFD+EF, high-fat diet with E. faecalis; SDHB, succinate dehydrogenase complex,subunit B; UCP, uncoupling protein; UQCRC, ubiquinol cytochrome c reductase core protein. E. faecalis administration reduces obesity beneficial effects were not significantly related to the difference E. faecalis is a gram-positive bacterium belonging to Lactoba- in food intake, physical activity, or RER (online supplementary cillales at the order level. E. faecalis is a common commensal figure 3A–C). Importantly, E. faecalis treatment did not affect complement to the healthy human gastrointestinal tract as well body weight gain at thermoneutrality (online supplementary as a leading cause of hospital-acquired infections.28 To investi- figure 3I). Thus, these results strongly indicated that E. faecalis gate the role of E. faecalis in obesity, we used HFD-fed mice as increased energy expenditure and reduced adiposity by acti- our model. Interestingly, we found a significant reduction both in vating BAT and beige fat formation. body weight gain and fat mass in the mice treated with E. faecalis (figure 3A,B). We next examined whether E. faecalis treatment MA is enriched in serum metabolites and its administration could affect energy metabolism. Notably, E. faecalis treatment reduces adiposity significantly increased the core body temperature when animals A potential mechanism by which E. faecalis influences the were exposed to a cold environment (4°C, 4 hours) (figure 3C,D). host energy metabolism would be via the secondary metabo- In addition, in the E. faecalis-treated mice, energy expenditure lites generated during microbial fermentation in the gut. It was increased significantly compared with that of the control mice reported that UCP1 is activated by LCFAs.29 Recently, it has been (figure 3E). Consistently, histological analysis revealed that the reported that LCFAs serve as substrates permanently attached sizes of lipid droplets in BAT from the E. faecalis-t reated mice to UCP1 that promote the transfer of H+ from the inner to the were much smaller than those of the control mice (figure 3F). outer mitochondrial membrane.16 Hence, it was of interest to Correspondingly, the protein expressions of UCP1 and OXPHOS know if E. faecalis treatment alters serum LCFAs. Accordingly, were significantly upregulated in BAT and Sub adipose tissue we performed an untargeted metabolomics analysis of LCFAs in of the E. faecalis-treated mice (figure 3G,H). Furthermore, E. the serum samples. Oral gavage of E. faecalis significantly altered faecalis treatment improved glucose homoeostasis and reduced a variety of LCFAs (figure 4A). To identify which of these LCFAs hepatic steatosis (online supplementary figure 3D–H). These 5 Quan L-H , et al. Gut 2019;0:1–9. doi:10.1136/gutjnl-2019-319114
Gut microbiota Gut: first published as 10.1136/gutjnl-2019-319114 on 19 November 2019. Downloaded from http://gut.bmj.com/ on November 20, 2019 at Karolinska BIBSAM Consortia. Protected by copyright. Figure 4 Myristoleic acid (MA) administration reduces adiposity. (A) E. faecalis (EF) treatment alters serum LCFAs in HFD-fed mice (n=9). (B) EF increases MA levels in MRS broth (n=6). (C) Oxygen consumption rates at day 6 of brown adipogenesis with 10 uM MA or BSA treatment (n=5–6). To investigate the potential effects of MA on whole-b ody energy metabolism, db/db mice were treated with MA (5 mg/kg) for 8 weeks by daily oral gavage (n=7–10). (D–E) MA treatment significantly decreased body weight gain as well as the fat mass of db/db mice. (F–G) Core body temperature and energy expenditure were significantly increased after MA treatment in db/db mice. (H) Representative H\&E staining of BAT from db/db mice. (I–J) MA treatment significantly increased UCP1 expression and mitochondrial OXPHOS protein expression in BAT and Sub of db/db mice. For body weight and seahorse oxygen consumption curves, statistical analysis was performed using a repeated measure two-w ay ANOVA, for energy expenditure, ANCOVA were employed and the rest of the statistics were performed with Student’s t-test. Data are means±SEM. *p<0.05, **p<0.01. ATP, adenosine triphosphate; BAT, brown adipose tissue; EP, epididymal fat; HFD, high-fat diet; LCFA, long-c hain fatty acid; SDHB, succinate dehydrogenase complex,subunit B; UCP, uncoupling protein; UQCRC, ubiquinol cytochrome c reductase core protein. were specifically altered after E. faecalis treatment, we further Moreover, MA significantly increased UCP1 expression (online analysed the amount of LCFAs in the culture medium with and supplementary figure 4B). These results implied that MA might without E. faecalis treatment. Intriguingly, we found a very increase the BAT activity. To further clarify this hypothesis, we high amount of MA (C14:1) in the E. faecalis treatment that used transgenic mice in which luciferase activity reveals endoge- reached a maximum of 12.3 times the amount found without nous UCP1 expression.30 Interestingly, the luciferase activity was the treatment (figure 4B). In addition, non-adecylic acid (C19:0) significantly increased on MA treatment (online supplementary and linoleic acid (C18:2) also showed some degree of significant figure 4C,D). These results evidenced the potential of the efficacy accumulation (5.9 and 4.3 times the level without the treatment, of MA against obesity. To test this hypothesis, db/db mice were respectively), whereas this accumulation pattern was found to a fed MA by oral gavage. As expected, MA significantly reduced lesser extent with other LCFAs (figure 4B). These results strongly body weight gain (figure 4D) and adiposity (figure 4E, online suggested that E. faecalis may be involved in fatty acid metabo- supplementary figure 4E) via increasing BAT thermogenesis lism. To further investigate the potential effect of LCFAs on BAT and whole-b ody energy metabolism (figure 4F,G, online supple- activity, primary brown adipocytes were treated with various mentary figure 4F). In addition, we revealed that MA activated LCFAs and cellular oxygen consumption was assessed. The results BAT (figure 4H,I), induced beige fat formation (figure 4J), and of the cellular experiment demonstrated that the top-ranking reversed hepatic steatosis (online supplementary figure 4J–K). metabolite, MA, significantly up-regulated oxygen consump- These beneficial effects did not significantly correlate with alter- tion (figure 4C). However, other fatty acids did not show such ations in either food intake, or physical activity, or RER (online effects, including linoleic acid (online supplementary figure 4A). supplementary figure 4G–I). To assess whether our results might 6 Quan L-H, et al. Gut 2019;0:1–9. doi:10.1136/gutjnl-2019-319114
be explained by the anti-o besity effect of MA on BAT, we used Gut microbiota Gut: first published as 10.1136/gutjnl-2019-319114 on 19 November 2019. Downloaded from http://gut.bmj.com/ on November 20, 2019 at Karolinska BIBSAM Consortia. Protected by UCP1 KO mice, which display greatly reduced BAT activity, to copyright. further verify our results. Accordingly, MA was able to reduce mediated by food intake or physical activity (online supplemen- the gain in body weight induced by an HFD in wild type (WT) tary figure 5I–J). Importantly, MA treatment did not affect body mice, but not in UCP1 KO mice (online supplementary figure weight gain at thermoneutrality (online supplementary figure 5A–C). In addition, MA could increase BAT thermogenesis 5Q). Taken together, these results demonstrated that E. faecalis- and improve glucose homoeostasis under HFD conditions in produced MA-reduced adiposity by increasing BAT activity and WT mice (due to hypothermia, the cold challenge experiment whole-body metabolism. was not performed on UCP1 KO mice) (online supplementary figure 5D–G). Additionally, energy expenditure increased in Reduction of E. faecalis MA production impairs anti-obesity the WT mice, but not in UCP1 KO mice (online supplementary effects figure 5H). Furthermore, MA improved BAT hypertrophy and The previous report demonstrated that ACOTs are a large reversed hepatic steatosis in WT mice, but not in the UCP1 KO family of enzymes that catalyse the hydrolysis of the thioester mice (online supplementary figure 5K, L, O, P). Accordingly, bond between a carbonyl group and a sulfur atom, producing the UCP1 protein expression was significantly up-regulated in fatty acids and CoA (figure 5A). In humans, Them1 thioesterase BAT and Sub of the MA treated WT mice (online supplementary hydrolyzes a range of fatty acyl-CoAs with a preference for long- figure 5M–N). Furthermore, these beneficial effects were not chain acyl-C oA molecules, and dimerisation is induced by fatty acyl-CoAs, coenzyme A (CoASH).31 To elucidate how E. faecalis Figure 5 Acyl-C oA thioesterase (ACOT) of the E. faecalis (EF) is a key enzyme involved in MA synthesis. (A) Synthesis pathway of free fatty acid by ACOT. (B) Successful knock down of ACOT gene in EF strain by Crispr-d Cas9 technique (n=4–5). (C) Changes of LCFAs biosynthesis by WT and ACOT KD EF in MRS broth (n=4). HFD-fed mice were treated daily with WT and ACOT KD EF (1×1010 CFU) by oral gavage for 5 weeks (n=6–8). (D–E) Body weight gain and fat mass were significantly decreased after being treated with the WT strain but not with the KD strain in HFD-fed mice. (F–G) Energy expenditure and core body temperature were significantly increased after WT strain but not KD strain treated in HFD-fed mice. (H–I) Endogenous UCP1 activity was significantly decreased after being treated with the WT strain but not the kD strain in HFD-fed mice (n=5). (J) Ginseng extract enriched EF which can produce LCFA, specifically MA by ACOT gene and this further reduced obesity by increasing bat activity and beige fat formation. Data are means±SEM. HFD, high-fat diet. For body weight curves, statistical analysis was performed using a repeated measure two-w ay ANOVA, for energy expenditure, ANCOVA were employed and the rest of the statistics was performed by one-w ay ANOVA with Tukey’s post-hoc tests. *p<0.05; **p<0.01; ***p<0.0001; NS, not statistically significant. BAT, brown adipose tissue; EP, epididymal fat; HFD, high-fat diet; HFD+WT EF, high- fat diet with WT E. faecalis; HFD+KD EF, high-fat diet with ACOT KD EF; KD, knock down; LCFA, long-chain fatty acid; UCP, uncoupling protein; WT, wild type. Quan L-H , et al. Gut 2019;0:1–9. doi:10.1136/gutjnl-2019-319114 7
Gut microbiota found that E. faecalis were significantly enriched at the family- Gut: first published as 10.1136/gutjnl-2019-319114 on 19 November 2019. Downloaded from http://gut.bmj.com/ on November 20, 2019 at Karolinska BIBSAM Consortia. Protected by genus-species levels after GE treatment. Furthermore, we found copyright. is involved in MA production, we first successfully established that E. faecalis could significantly reduce obesity by increasing ACOT gene knockdown (KD) and WT E. faecalis strain using BAT activity and beige fat formation. Interestingly, previous the Crispr-dCas9 technique (about 60% KD compared with reports demonstrated that heat-treated E. faecalis (bacteria that WT) (figure 5B). KD of ACOT gene did not alter the growth of were heated at 110°C for 10 min) had anti-o besity effects and E. faecalis (data not shown). Next, the WT and KD E. faecalis improved hepatic steatosis in DIO mice model.38 A study reports strains were incubated in a De Man, Rogosa and Sharpe (MRS) that autoclaving Akkermansia muciniphila eliminated the anti- culture medium, and LCFAs were subjected to an untargeted obesity effects of the live strain.39 In line with these observations, metabolomics analysis. Consistently, the WT strain was able to presumably, E. faecalis were biologically inactive after heat treat- induce MA accumulation, whereas the ACOT KD strain could ment, even though there is a lack of knowledge of the molecular not induce as much accumulation of MA as the WT strain mechanisms involved. (figure 5C). In addition, KD of ACOT gene did not significantly alter other LCFAs or SCFAs compared with the WT E. faecalis To understand the mechanisms of action, we speculated that (online supplementary figure 6A). These results clearly indicated the secondary metabolites that were produced during fermenta- that the production of MA is at least partly mediated by ACOT. tion could play important roles in BAT activation. Interestingly, To investigate the influence of genetically modified E. faecalis there are reports demonstrating that rats treated with SCFA, strains on UCP1 activity, a variety of mouse models were used. such as propionate and butyrate, showed significantly less gain in We observed that WT E. faecalis but not KD E. faecalis could body weight than that seen in untreated rats, despite similar food reduce adiposity in HFD-fed mice (figure 5D,E, online supple- intake.40 However, another study indicates that acetate promotes mentary figure 6B). Accordingly, WT but not KD E. faecalis was metabolic syndromes via the microbiome-brain-β-cell axis.41 able to increase cold-induced thermogenesis (figure 5G, online Surprisingly, in our experiment, SCFAs showed minimal or no supplementary figure 6C) and energy expenditure (figure 5F) differences after E. faecalis treatment (online supplementary without significant changes in food intake (online supplemen- figure 6A). Interestingly, we found that LCFA (MA) production tary figure 6D). Endogenous UCP1 activity was also significantly was exclusively elevated after E. faecalis treatment. Further- upregulated in WT strain but not with the KD E. faecalis treat- more, fatty acids 19:0 and 18:2 also showed some degrees of ment in the UCP1 luciferase transgenic mice model (figure 5H,I). increase after E. faecalis treatment (figure 4B). Recently, it has To avoid interspecies crosstalk between gut microbiota, mice been reported that LCFAs serve as permanently attached UCP1 were pretreated with ABX. Consistently, we found that the WT substrates that help to carry H+ via UCP1.16 The results of strain could increase BAT activity and cold-induced thermogen- the oxygen consumption experiment indicated that C19:0 and esis, whereas, the KD strain-treated db/db mice could not (online C18:2 have minor effects compared with those of C14:1 (online supplementary figure 6E–F). These results clearly demonstrated supplementary figure 4A). These results highlighted the fact that the important role of the ACOT gene in MA biosynthesis. Taken E. faecalis induces MA levels, thereby involving UCP1-mediated together, we showed that E. faecalis drove MA production via heat generation. Detailed molecular analysis revealed that the E. the ACOT gene. faecalis-encoding ACOT gene was responsible for the biosyn- thesis of MA. In addition, in the treatment of the ACOT gene KD, Discussion E. faecalis was unable to reduce the body weight gain compared Although it has been shown that as a TCM, ginseng displays with that in the WT E. faecalis in HFD-fed mice. However, data various biological functions including effects against obesity and on the types of substrates (eg, SCFAs or carbohydrates) that obesity-related diseases, the underlying molecular mechanism is favour MA production are still largely unknown. Besides, our still unclear. In this study, by integrating data on the gut micro- research does not rule out other possible alternative mechanisms biome, serum metabolome, and BAT, we demonstrated that the of the anti-o besity effects of ginseng, including reduced adipo- GE–E. faecalis−LCFA (specifically MA) axis reduces obesity by cyte hypertrophy via the modulation of angiogenesis and MMP increasing BAT activity and beige fat formation. Moreover, E. activity,42 increased energy expenditure via stimulation of the faecalis was observed to drive the MA production via the ACOT adenosine monophosphate-activated kinase pathway,20 reduced gene. In addition, we demonstrated for the first time in this adipogenesis and inflammation,43 and maintenance of the gut study that as an LCFA, E. faecalis-p roduced MA has tremen- barrier integrity.44 dous potential for treating metabolic syndrome disorders, such as obesity (figure 5J). In summary, by integrating data on the gut microbiome, serum metabolome, and BAT, we demonstrated that the GE–E. faecalis– Obesity is a chronic medical condition, which develops due to LCFA (specifically MA) axis reduces obesity by increasing BAT many factors, including genetic factors, lifestyle, diet, gut micro- activity and beige fat formation. In addition, we observed that biota, and unbalanced energy metabolism. Therefore, drugs that E. faecalis drives MA production via the ACOT gene. Further- aim to increase energy expenditure have the potential to be the more, we demonstrated for the first time that as an LCFA, E. most effective and attractive strategy against obesity.32 As an faecalis-produced MA has tremendous potential to treat meta- energy-consuming organ, BAT has received much attention.33 bolic syndrome disorders, such as obesity. Therefore, increasing BAT mass and/or activity is a promising strategy to treat obesity and metabolic diseases. Indeed, studies Author affiliations by our group and others have shown that BAT transplantation 1Key Laboratory of Natural Resource of the Changbai Mountain and Functional or the use of rutin, which is able to activate BAT, could reverse Molecular, Ministry of Education, Agricultural College, Yanbian University, Yanji, China metabolic disorders in various obese mouse models.34–37 2Key Laboratory of Animal Ecology and Conservation Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, China Recently, several reports have pointed to the interaction 3Savaid Medical School, University of the Chinese Academy of Sciences, Beijing, between BAT and intestinal flora.10 These results suggest that China GE-mediated brown fat activation might be associated with 4School of Pharmaceutical Sciences, Zhengzhou University, Zhengzhou, China changes in the intestinal flora. Additionally, GE could also 5Institute of Neuroscience and Translational Medicine, College of Life Science and change the gut microbiota composition.23 Accordingly, we Agronomy, Zhoukou Normal University, Zhoukou, Henan, China 8 Quan L-H , et al. Gut 2019;0:1–9. doi:10.1136/gutjnl-2019-319114
6Jilin Provincial Key Laboratory of Transgenic Animal and Embryo Engineering, Gut microbiota Gut: first published as 10.1136/gutjnl-2019-319114 on 19 November 2019. Downloaded from http://gut.bmj.com/ on November 20, 2019 at Karolinska BIBSAM Consortia. Protected by Yanbian University, Yanji, China copyright. 7State Key Laboratory of Molecular Developmental Biology, Institute of Genetics and 17 Chang C-J, Lin C-S, Lu C-C, et al. Corrigendum: Ganoderma lucidum reduces Developmental Biology, Chinese Academy of Sciences, Beijing, China obesity in mice by modulating the composition of the gut microbiota. Nat Commun 2017;8:16130. Acknowledgements The authors would like to thank Dr Dangsheng Li for his great advices. 18 Kiefer D, Pantuso T. Panax ginseng. Am Fam Physician 2003;68:1539–42. 19 Patel S, Rauf A. Adaptogenic herb ginseng (Panax) as medical food: status quo and Contributors WJ, L-HQ, CZ, MD and DL. designed the research and analysed all of the results; L-HQ, JJ, HX and CY performed animal studies; CZ performed cellular future prospects. Biomed Pharmacother 2017;85:120–7. experiments and animal tissue analysis; MD performed animal tissue analysis and 20 Li Z, Ji GE. Ginseng and obesity. J Ginseng Res 2018;42:1–8. bacteria dCas9 construction; XL, HZhou, HZhang, LC, Z-BL and F-LZ performed 21 Dong W-W , Han X-Z , Zhao J, et al. Metabolite profiling of ginsenosides in rat plasma, animal tissue analysis and whole-b ody animal imaging; S-ML and GS performed lipidomics analysis; and L-HQ, CZ, MD and WJ wrote the paper. urine and feces by LC-MS/MS and its application to a pharmacokinetic study after oral administration of Panax ginseng extract. Biomed Chromatogr 2018;32. doi:10.1002/ Funding This work was supported by the strategic priority research program bmc.4105 (XDB13030000 to WJ), National Natural Science Foundation of China (31171131 2 2 Dong W-W, Xuan F-L, Zhong F-L, et al. Comparative analysis of the rats’ gut and 81370951 to WJ, 81660643 to LQ), National Key Research and Development microbiota composition in animals with different ginsenosides metabolizing activity. J Program of China (2017YFC1001003 to WJ). Agric Food Chem 2017;65:327–37. 2 3 Sun Y, Chen S, Wei R, et al. Metabolome and gut microbiota variation with long-term Competing interests None declared. intake of Panax ginseng extracts on rats. Food Funct 2018;9:3547–56. 2 4 Meyer CW, Willershäuser M, Jastroch M, et al. Adaptive thermogenesis and thermal Patient consent for publication Not required. conductance in wild-type and UCP1-K O mice. Am J Physiol Regul Integr Comp Physiol 2010;299:R1396–406. 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