Leuconostoc mesenteroides mediates an electrogenic pathway to attenuate the accumulation of abdominal fat mass induced by high fat diet

Although several electrogenic bacteria have been identified, the physiological effect of electricity generated by bacteria on host health remains elusive. We found that probiotic Leuconostoc mesenteroides (L. mesenteroides) can metabolize linoleic acid to yield electricity via an intracellular cyclophilin A-dependent pathway. Inhibition of cyclophilin A significantly abolished bacterial electricity and lowered the adhesion of L. mesenteroides to the human gut epithelial cell line. Butyrate from L. mesenteroides in the presence of linoleic acid were detectable and mediated free fatty acid receptor 2 (Ffar2) to reduce the lipid contents in differentiating 3T3-L1 adipocytes. Oral administration of L. mesenteroides plus linoleic acid remarkably reduced high-fat-diet (HFD)-induced formation of 4-hydroxy-2-nonenal (4-HNE), a reactive oxygen species (ROS) biomarker, and decreased abdominal fat mass in mice. The reduction of 4-HNE and abdominal fat mass was reversed when cyclophilin A inhibitor-pretreated bacteria were administered to mice. Our studies present a novel mechanism of reducing abdominal fat mass by electrogenic L. mesenteroides which may yield electrons to enhance colonization and sustain high amounts of butyrate to limit ROS during adipocyte differentiation.


Electricity detection.
A lab-fabricated chamber equipped with a cathode and anode was used for the in vitro detection of bacterial electricity. A carbon filth (2.5 cm × 10 cm) (Homy Tech, Taoyuan, Taiwan) served as an anode. A carbon cloth (10 cm × 10 cm) (Homy Tech) covered with a nafion (sulfonated tetrafluoroethylene based fluoropolymer-copolymer) membrane N117 (6 cm × 6 cm) (Homy Tech), a proton exchange membrane (PEM), functioned as a cathode. Anode and cathode were connected by copper wires, which sequentially were attached to 1000 Ω external resistance. L. mesenteroides EH-1 bacteria [10 7 colony-forming unit (CFU)/ml] in rich media [10 g/l yeast extract (Biokar Diagnostics, Beauvais, France), 5 g/l TSB, 2.5 g/l K 2 HPO 4 and 1.5 g/l KH 2 PO 4 ] in the absence of presence of 2% linoleic acid were pipetted on the surface of the anode. L. mesenteroides EH-1 in rich media was pretreated with 2 µM TMN355 (Santa Cruz Biotechnology, Dallas, TX, USA), an inhibitor of cyclophilin A dissolved in 2% final concentration of dimethyl sulfoxide (DMSO), at 37 °C for 24 h followed by washing twice with 1 × PBS. The changes in voltage (mV) against time (min) were recorded by a digital multimeter (Lutron, DM-9962SD, Sydney, Australia). The voltage was recorded every 10 s to plot a graph of voltage against time.

Real-time polymerase chain reaction (PCR). A StepOnePlus Real-time PCR System (ThermoFisher
Scientific, Waltham, MA, USA) using Power SYBR Green and PCR Master Mix (ThermoFisher Scientific) was used to examine gene expression of the cyclophilin A in L. mesenteroides EH-1 treated with/without 2 μM TMN355 (Bio-Techne Corporation, Minneapolis, MN, USA). RNA (1 ng) was converted into cDNA using an iScript cDNA Synthesis kit (Bio-Rad, Hercules, CA, USA). cDNA (50 ng/μl) of L. mesenteroides EH-1 was used as a template. Primers were designed using the Primer-Blast tool from the National Center for Biotechnology Information (NCBI). The reaction conditions were set for 40 cycles as follows: 95 °C for 10 min followed by 95 °C for 15 s, 48 °C for 60 s, and 72 °C for 30 s. A complete reaction was achieved with three biological replicates, and each sample consisted of three technical replicates. The gene expression of triosephosphate isomerase (tpi) was used for normalization. The relative expression levels were analyzed using the cycle threshold (2 −ΔΔCt ) method. Primers included forward 5′ TCC AAA CTA GGA TAG CCG CC 3′ and reverse 5′ TTC GTG GCG CTG TTT CAA TG 3′ for cyclophilin A; and forward 5′ ACC CTC AGT GGC TCA AGT GG 3′ and reverse 5′ GGC CAG CGT CTG ACG TAT CA 3′ for tpi.
Gas chromatography mass spectrometry (GC-MS) analysis. Leuconostoc mesenteroides EH-1 (10 7 CFU/ml) was incubated in 10 ml rich media in the presence of 2% linoleic acid for 24 h. After centrifugation at 5000 × g for 10 min, bacteria in supernatants were further removed by 0.22 µm filters. SCFAs in the fermentation media were detected by ethyl acetate (Residue Analysis OmniSolv, EMD Millipore, Billerica, MA) liquid-liquid extraction after addition of 50 µl of 2 H 7 -butyrate (1 mg/ml) (C/D/N Isotopes, Quebec, Canada) as an internal standard, acidification with 0.5% ortho-phosphoric acid (ThermoFisher Scientific) and saturation with sodium chloride (ThermoFisher Scientific) followed by GC-MS analysis using an Agilent 5890 Series II GC in conjunction with 5971 MS detector (Agilent Technologies, Inc., Palo Alto, CA). A 70 eV electron was utilized for ionization. The levels of SCFAs in the fermentation media were quantified by a calibration curve made from six non-zero levels using the Free Fatty Acids Test Standard (Restek Corporation, Bellefonte, PA) which was diluted 500-, 1000-, 2000-, 5000-, and 10,000-folds.

Results
Electricity and SCFAs were produced by L. mesenteroides EH-1 plus linoleic acid. In the presence of a variety carbon sources, several probiotic bacteria are able to yield acetate and butyrate which are known to be electron donors in a microbial fuel cell system [33][34][35] . We thus examined the eletrogenicity of probiotic L. mesenteroides EH-1 strain in the presence of 2% linoleic acid as a carbon source. An in vitro chamber with cathode and anode electrodes was fabricated to detect bacterially generated electricity. As shown in Fig. 1a, little or no voltage change was recorded over a monitoring period of 70 min in media with linoleic acid alone. A slight increase in voltage was detected in the media with L. mesenteroides EH-1 alone. The voltage was considerably raised to a peak of more than 1 mV when bacteria were placed in media in the presence of 2% linoleic acid. These data demonstrate that L. mesenteroides EH-1 is an electrogenic bacterium. We next examined the effect of bacterial electricity on regulation of the redox cycling of iron. In a ferrozine assay, linoleic acid, L. mesenteroides EH-1 or L. mesenteroides EH-1 plus linoleic acid were added into a reaction solution containing FMN, ferrozine and ferric (Fe 3+ ) ammonium citrate. As shown in Fig. 1b,c, the concentration of ferrozine-chelatable iron (dark brown complex) in the reaction solution containing L. mesenteroides EH-1 plus linoleic acid was markedly higher than in the reaction solution containing linoleic acid alone or bacteria alone. This result suggests that electrons produced by L. mesenteroides EH-1 plus linoleic acid converted Fe 3+ to ferrozine-chelatable iron. To determine whether SCFAs were produced in the culture of L. mesenteroides EH-1 plus linoleic acid, L. mesenteroides EH-1 was cultured in rich media in the presence of 2% linoleic acid for 24 h. Rich media with linoleic acid alone or L. mesenteroides EH-1 alone served as controls. The media in the culture of L. mesenteroides EH-1 with linoleic acid turned yellow after incubation for 24 h (Fig. 1d). As shown in Fig. 1c, the OD 562 of media with L. mesenteroides EH-1 plus linoleic acid demonstrated significant decreases compared to controls, indicating that linoleic acid was fermented by L. mesenteroides EH-1. GC-MS analysis was performed to quantify the level of SCFAs in fermentation media of L. mesenteroides EH-1. Nine SCFAs including acetate, propionate, and butyrate were detectable in media from linoleic acid fermentation of L. mesenteroides EH-1 (Fig. 1f).

Adipocyte differentiation was attenuated by fermentation media of L. mesenteroides and butyrate.
To explore if linoleic acid fermentation of L. mesenteroides EH-1 affects adipocyte differentiation, we added the supernatant from the culture of L. mesenteroides EH-1 plus linoleic acid onto the differentiating 3T3-L1 preadipocytes. Lipid accumulation during 3T3-L1 differentiation were detected by Oil Red O staining. The content of lipids was significantly increased during cell differentiation when the culture media of 3T3-L1 preadipocytes were replaced with differentiation media (Fig. 2a). The differentiation-induced increase in lipids were markedly inhibited by addition of supernatant of the culture of L. mesenteroides EH-1 plus linoleic acid. There was no change in lipid content in differentiated 3T3-L1 cells after addition of media containing linoleic acid alone, although inhibition of lipid production was observed by adding the supernatant of the culture of L. mesenteroides EH-1 alone. To examine the contribution of SCFAs to adipocyte differentiation, the differentiated 3T3-L1 cells treated with supernatant of the culture of bacteria plus linoleic acid were added with GLPG-0974, a free fatty acid receptor 2 (Ffar2) antagonist. Inhibition of Ffar2 by GLPG-0974, not its DMSO solvent, significantly diminished the action of supernatant of the culture of bacteria plus linoleic acid on the inhibition of lipid production (Fig. 2b). Furthermore, cells treated with butyrate markedly reduced lipid contents. The reduction can be reversed by addition of GLPG-0974. Results above clearly demonstrated that Ffar2 mediated the effect of butyrate, one of SCFAs produced by linoleic acid fermentation of L. mesenteroides EH-1 alone, on adipocyte differentiation. www.nature.com/scientificreports/ differentiating cells was greatly suppressed by addition of supernatant of the culture of L. mesenteroides EH-1 plus linoleic acid, although suppression of ROS production was also detected by adding the supernatant of the culture of linoleic acid or L. mesenteroides EH-1 alone (Fig. 3a).

The formation of ROS and 4-HNE was suppressed by L. mesenteroides
To evaluate the ability of L. mesenteroides EH-1 to attenuate ROS production in vivo, we examined the formation of 4-HNE, a secondary product of oxidative stress 37 , in abdominal fat pads of ICR mice fed with SCD or HFD. The level of 4-HNE detected by western blotting in HFD-fed mice was significantly higher than that in SCD-fed mice (Fig. 3b). The level of 4-HNE remained high in mice fed a HFD supplemented with linoleic acid (Fig. 3b). By contrast, the high level of 4-HNE in abdominal fat of HFD-fed mice was markedly reduced when mice were co-administered L. mesenteroides EH-1 bacteria alone or bacteria plus linoleic acid by oral gavage (Fig. 3b). Results from high-performance liquid chromatography (HPLC) analysis (Fig. S3) showed that butyrate of greater than 1 mmol/l was detected in cecum of HFD-fed mice administered with L. mesenteroides EH-1 alone or plus linoleic acid (Fig. S3). The result suggested butyrate produced by L. mesenteroides EH-1 may down-regulate the formation of oxidative stress in differentiating adipocytes and abdominal fat depots.
Cyclophilin A mediated electricity production of L. mesenteroides EH-1. FMN-based extracellular electron transfer (EET) is a process of electricity production in Gram-positive bacteria which express peptide pheromone-encoding lipoprotein A on the bacterial membrane. This binds two flavin molecules, enabling electrons to exit the membrane to reach the bacteria's exterior [38][39][40] . Although several intracellular molecules or membrane proteins in bacteria function as electron donors or acceptors, respectively, the mediators that transport the electrons from donors to acceptors are not well characterized. Cyclophilin A can sequester cytochrome C, an electron carrier protein 41 . Furthermore, it can bind to peroxiredoxin proteins to support its peroxidase www.nature.com/scientificreports/ www.nature.com/scientificreports/ activity as an immediate electron donor 25 . When L. mesenteroides EH-1 was pretreated for 24 h with TMN355, a potent cyclophilin A inhibitor, gene expression of cyclophilin A was significantly reduced by about 50% (Fig. 4a). TMN355 itself did not affect the growth of L. mesenteroides EH-1 (Fig. S2). To examine whether reduction of cyclophilin A expression influences bacterial electron production, L. mesenteroides EH-1 bacteria pretreated with TMN355 were added into media supplemented with 2% linoleic acid in an in vitro chamber with cathode and anode electrodes. Pretreatment of L. mesenteroides EH-1 with TMN355 led to a marked attenuation of voltage production relative to bacteria without TMN355 pretreatment (Fig. 4b). This result indicated that cyclophilin A mediated the electricity production of L. mesenteroides EH-1. The high concentration of ferrozine-chelatable iron was considerably reduced when the reaction solution contained TMN355-pretreated L. mesenteroides EH-1 plus linoleic acid. This result illustrated that L. mesenteroides EH-1 plus linoleic acid promoted the reduction

Cyclophilin A was essential for bacterial adhesion and reduction of the formation of 4-HNE and abdominal fat depots. A human epithelial cell line Caco-2, a widely used model of the intestinal epithelial
barrier, was ultilized to access whether the electron produced by L. mesenteroides EH-1 influenced the bacterial adhesion. Pretreatment of L. mesenteroides EH-1 with TMN355 resulted in a significant decrease in the number of bacteria adhered to Caco-2 cell (Fig. 4e,f). We next examined whether inhibition of cyclophilin A altered the ability of L. mesenteroides EH-1 to mitigate ROS production in vivo. As shown in Fig. 4g, the level of 4-HNE in abdominal fat of HFD-fed mice administered with TMN355-pretreated L. mesenteroides EH-1 plus linoleic acid was noticeably higher than mice administered with L. mesenteroides EH-1 plus linoleic acid. Since inhibition of cyclophilin A by TMN355 diminished the electron production, the cyclophilin A-mediated electron production may play a function role in the regulation of bacterial attachment to gut epithelia barrier. In Fig. S3, we have demonstrated that a high amount (> 1.5 mmol/l) of butyrate was produced in cecum of mice administered with L. mesenteroides EH-1 plus linoleic acid. Thus, electron mediated by cyclophilin A may facilitate the bacterial adhesion to sustain the high amounts of butyrate for reduction of 4-HNE formation in abdominal fat. We next investigated the consequence of cyclophilin A-mediated electricity on the accumulation of abdominal fat mass in HFD-fed mice. Compared to mice fed with SCD, mice fed with HFD exhibited markedly increased abdominal fat mass (Fig. 5a,c) and body weight (Fig. 5b). Obesity with high body weight was observed in HFDfed mice receiving orally-administered linoleic acid alone (Fig. 5d,e). Administration of L. mesenteroides EH-1 alone, however, caused a reduction of abdominal fat mass (Fig. 5d,f) and body weight (Fig. 5e). This reduction www.nature.com/scientificreports/ was significantly enhanced when mice were administered L. mesenteroides EH-1 plus linoleic acid, resulting in the body weight and fat accumulation similar to that of mice fed with SCD. The reduction of body weight and abdominal fat mass was markedly reduced when mice were treated with TMN355-pretreated L. mesenteroides EH-1 plus linoleic acid ( Fig. 5d-f). Taken together, results from Figs. 4 and 5 indicated that cyclophilin A-dependent electron generation by L. mesenteroides EH-1 regulated lipid accumulation during adipogenesis.

Discussion
While the electricity produced by L. mesenteroides EH-1 is readily detectable in the presence of 2% linoleic acid (Fig. 1a), a low but detectable voltage change was also observed in TSB media containing L. mesenteroides EH-1 without addition of linoleic acid. One possible explanation for this detectable electricity is the presence of dextrose in TSB which serves as a potential elecrogenic carbon source. Similarly, although linoleic acid augmented the suppressive effect of L. mesenteroides EH-1 on differentiation-induced increase of ROS (Fig. 3a) and lipids in vitro (Fig. 2a) and HFD-induced 4-HNE (Fig. 3b) and abdominal fat masses in vivo (Fig. 5), L. mesenteroides EH-1 alone without linoleic acid still can induce some of the same suppressive effects but to a lesser extent. In the absence of linoleic acid, L. mesenteroides EH-1 may generate electricity by using other carbon sources for fermentation L. mesenteroides EH-1, such as carbohydrates in culture media or the mouse gut. When mice fed with HFD, carbohydrates in HFD can be converted to monosaccharides of glucose, fructose and galactose 44,45 which will be carbon sources for L. mesenteroides EH-1 fermentation to produce electricity and SCFAs. Gram-positive strains of Lactobacillus, Propionibacterium, and Bifidobacterium bacteria metabolize linoleic acid to vaccenic acid, 10-hydroxy-18:1, and conjugated linoleic acid as a final product which has been found to improve human health 30 . Our results (Fig. 5d-f) demonstrated that feeding mice with linoleic acid alone did not prevent the formation of 4-HNE and abdominal fat masses, suggesting that linoleic acid and its metabolites generated by mouse cells have no effects on adipogenesis. Bacterial fermentation products such as acetate, butyrate, and ethanol can be electron donors [33][34][35] . It has been reported that acetate and butyrate can attenuate lipopolysaccharide (LPS)-induced lipid peroxidation and ROS 46,47 . Our data demonstrated that L. mesenteroides EH-1 used linoleic acid as a carbon source to undergo fermentation (Fig. 1e,f) and produced SCFAs such as acetate and butyrate (Fig. S3). In our previous study, butyrate generated from glucose fermentation by L. mesenteroides EH-1 maintained glucose level and enhanced insulin sensitivity 28 . In this study, we have screened the supernatant following linoleic acid fermentation of L. mesenteroides (Fig. 1d,e) to quantify their butyrate producing capacity of 0.05 mmol/l by GC-MS analysis (Fig. 1f). However, a higher concentration of butyrate www.nature.com/scientificreports/ in mice gut was detected as a sharp specific peak in the HPLC chromatogram and was determined to be at a concentration of > 1.5 mmol/l in the linoleic acid fermented media by comparison to a butyrate standard curve (Fig. S3). Blockade of cyclophilin A in L. mesenteroides EH-1 by TMN355 limited the electricity production and bacterial adhesion to epithelial cell line Caco-2 (Fig. 4). We envision that bacteria produced electron to enhance their colonization on gut epithelial barrier which can lead to the maximum yield of butyrate in the gut. Butyrate may reach adipocytes in abdominal fats via the bloodstream to regulate the accumulation of 4-HNE and fat mass during adipogenesis (Fig. 6). Cyclophilins are expressed in many tissues and cellular compartments where they act as chaperones to assist protein folding and interaction 48,49 . It has been acknowledged that diverse organisms increase the expression of cyclophilin genes as a defense against oxidative stress 50 . Moreover, cyclophilins can stimulate their antioxidant activity by binding and donating electrons to antioxidant enzymes 25 . Our data demonstrate for the first time that TMN355 down-regulated the expression of cyclophilin A and blocked electricity production (Fig. 4a-d), highlighting the essential role of cyclophilin A in the EET system of L. mesenteroides EH-1. Addition of 0.5 mmol/l FMN to the culture of L. mesenteroides EH-1 plus linoleic acid significantly enhanced bacterial electricity production (Fig. S1), suggesting L. mesenteroides EH-1, as other Gram-positive bacteria, utilize the FMN-based EET www.nature.com/scientificreports/ system 51 to yield electricity. Future work will investigate the engagement among cyclophilin A, FMN and other components in the EET system of L. mesenteroides EH-1. Electrons generated by bacterial fermentation are involved in a range of physiological functions 19 . For example, electrons can enhance NAD(P)H and flavoprotein expression, collapse the rate of ROS production, and modulate cell metabolism 52 . Moreover, the role of bioelectricity in the intestinal epithelium has been determined to attract various cells 53 . Interestingly, the extent of the ROS response to enforced electrons may depend on spin-mixing of orbital electron spins with opposite adjacent electron, resulting in a decrease of electrochemical potential 54 . ROS has previously been found to stimulate lipid accumulation during adipocyte differentiation from preadipocytes 55 . It has been reported that the Gram-positive bacteria in mouse gut can mediate EET to produce electricity 19 . Although we cannot exclude the possibility that electrons produced in the gut can travel in the bloodstream to abdominal adipocytes to control adipogenesis, our data demonstrated that butyrate was produced in cecum of mice administered with L. mesenteroides EH-1 plus linoleic acid. Butyrate may reach the abdominal adipocytes via bloodstream and eliminate accumulated ROS in differentiated adipocytes. Metabolites such as glutathione (GSH) and SCFAs produced by gut bacteria have been largely recognized to modulate oxidizing conditions toward adipogenesis in adipose tissues 42,56,57 . Our results revealed that electrogenic L. mesenteroides EH-1 is a probiotic candidate for suppression of ROS-associated accumulation of abdominal fat mass.
Elevated lipid levels and oxidative stress are the primary pathological processes underlying obesity-related disease. The regulation of cyclophilin A-mediated electricity production in L. mesenteroides EH-1 helps alleviate ROS in abdominal adipocytes, successfully ameliorating HFD-induced abdominal fat deposition. Thus, the suppressive effect of L. mesenteroides EH-1 on the accumulation of abdominal fat masses can be achieved by eradicating ROS through a novel mechanism associated with butyrate in fermentation production and cyclophilin A-mediated electron production. Although electrogenic bacteria in the gut have been identified 58 and can be used to predict lymphocyte recruitment 53 , we demonstrate here for the first time that L. mesenteroides EH-1 benefits human health by reduction of HFD-induced accumulation of abdominal fat mass.