Lactobacillus reuteri ZJ617 attenuates metabolic syndrome via microbiota-derived spermidine

Ma, Yanfei; Zhong, Yifan; Tang, Wenjie; Valencak, Teresa G.; Liu, Jingliang; Deng, Zhaoxi; Mao, Jiangdi; Liu, Daren; Wang, Shanshan; Wang, Yuhao; Wang, Haifeng

doi:10.1038/s41467-025-56105-4

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Published: 21 January 2025

Lactobacillus reuteri ZJ617 attenuates metabolic syndrome via microbiota-derived spermidine

Nature Communications volume 16, Article number: 877 (2025) Cite this article

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Abstract

Metabolic syndrome (MetS) is a difficult-to-manage disease that poses a significant risk to human health. Here, we show that the supplementation of Lactobacillus reuteri ZJ617 ameliorates symptoms of MetS in mice induced by the high-fat diet. L. reuteri ZJ617 modulates host metabolism by interacting with the microbiome, resulting in the production of spermidine synthesized by the microbiota. L. reuteri ZJ617 serves as a source of substrates for the microbiota to synthesize spermidine, hence preventing the decline of bacteria responsible for spermidine production. Spermidine treatment mimics the metabolic effects of L. reuteri ZJ617, whereas pharmacological inhibition of spermidine biosynthesis in mice abolishes these benefits. Our findings reveal the mechanism by which L. reuteri ZJ617 alleviates MetS symptoms and provide support for its potential use as a probiotic for promoting metabolic health.

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Introduction

The metabolic syndrome (MetS) is a prevalent global health issue. If left untreated, MetS may lead to deleterious metabolic illnesses, including obesity, type 2 diabetes mellitus (T2D), and nonalcoholic fatty liver disease (NAFLD)^1,2. In mammals, adipose tissue is an active endocrine metabolic organ involving whole-body energy homeostasis³. Individuals with an excess of intra-abdominal or visceral adipose tissue are at substantially higher risk of being diagnosed with MetS⁴. Although white adipose tissue (WAT) acts to store energy, brown adipose tissue (BAT), which is limited in adult humans^5,6, plays a crucial role in energy expenditure⁷. Therefore, transforming white to brown-like adipocytes are a promising strategy for treating metabolic disorders.

Lactobacillus species have a long history of safe use in food and have been proven to improve host metabolic health⁸. Shotgun metagenomic sequencing has revealed a positive correlation between L. reuteri in feces and reduced adiposity⁹. Multiple randomized clinical trials have reported an inverse relationship between the consumption of L. reuteri and the incidence of MetS^10,11,12. A prior study also demonstrated an anti-obesity effect of L. reuteri, which appears to be associated with the remodeling of WAT and energy metabolism¹³. However, the molecular mechanism underlying the metabolic benefits of L. reuteri requires further clarification.

In the intestine, trillions of commensal bacteria produce a variety of bioactive metabolites and play essential roles in metabolism¹⁴. These metabolites, including short-chain fatty acids, bile acids, branched-chain amino acids, indole derivatives, and endocannabinoids, can reprogram energy expenditure by affecting non-shivering thermogenesis in adipose tissue^15,16. Given that a recent study revealed a close link between the metabolic regulatory ability of Lactobacillus and the intestinal microbiota¹⁷, it is possible that L. reuteri delivers metabolic benefits to the host by promoting the production of certain metabolites from the microbiota. Thus, the goal of the present study was to gain a deeper understanding of the beneficial effects of L. reuteri in relation to metabolic syndrome and to determine the potential role of gut microbiota and their metabolites in achieving these benefits.

Our previous studies have demonstrated that dietary supplementation with L. reuteri ZJ617 improves the health of the intestine and liver^18,19,20. The present study further revealed that L. reuteri ZJ617 alleviates MetS and promotes the transforming of white to brown-like adipose tissue in mice fed with a high-fat diet (HFD). Mechanistically, L. reuteri ZJ617 provides substrates to spermidine-producing bacteria, promoting the production of spermidine and preserving sufficient levels of spermidine in circulation and WAT. Spermidine supplementation delivers similar metabolic benefits to L. reuteri ZJ617, while pharmaceutical inhibition of spermidine biosynthesis abolished these benefits. Our findings highlight the significance of spermidine as a microbiota-derived molecule that contributes to the metabolic benefits of L. reuteri and further supports the use of L. reuteri as a probiotic to treat MetS.

Results

L. reuteri ZJ617 alleviates the symptoms of metabolic syndrome in HFD-fed mice

In order to evaluate the effects of L. reuteri ZJ617 on MetS, we chose HFD-fed mice as the model (Fig. 1a). As expected, the mice fed the HFD for 14 weeks developed obesity (Fig. 1b, c) and exhibited reduced oxygen consumption, CO₂ production, and energy expenditure in both light and dark (Supplementary Fig. 1a, b) in comparison to the control group of mice fed a low-fat diet (LFD). However, supplementing HFD-fed mice with L. reuteri ZJ617 effectively reduced body weight gain (Fig. 1b, c), improved oxygen consumption, CO₂ production, energy expenditure (Fig. 1d and Supplementary Fig. 1c), and reversed the decrease of rectal temperature caused by HFD (Fig. 1e). In addition, L. reuteri ZJ617 supplementation in HFD-fed mice effectively reduced the concentrations of serum triglycerides (TG), total cholesterol (TC) and low-density lipoprotein cholesterol (LDL-C) (Supplementary Fig. 1d–f), and efficiently improved glucose tolerance and insulin sensitivity (Fig. 1f, g). The homeostasis model assessment of insulin resistance (HOMA-IR) score further revealed that L. reuteri ZJ617 supplementation was able to attenuate the severity of insulin resistance caused by HFD (Fig. 1h). Importantly, the comparable liver weight, alleviated hepatic steatosis (Supplementary Fig. 1g–i), and improved liver function (Supplementary Fig. 1j, k) demonstrate that long-term oral administration of L. reuteri ZJ617 had limited side effects. Of note, there was no significant difference in food intake between mice supplemented with L.reuteri ZJ617 and mice fed with only HFD (Supplementary Fig. 1l, m), suggesting that the metabolic benefits of L. reuteri ZJ617 were not due to a decrease in food consumption.

**Fig. 1: *L. reuteri* ZJ617 alleviates the feature of metabolic syndrome in HFD-fed mice.**

Remarkably, detailed tissue analysis showed that mice supplemented with L.reuteri ZJ617 had significantly fewer adipose depots and smaller lipid droplets in adipocytes than mice fed with only HFD (Fig. 1i–k). Transmission electron microscopy (TEM) showed a higher density of mitochondria (Fig. 1l and Supplementary Fig. 1n) and higher expression of mitochondrial biogenesis genes (Cox5b, Cox7a1, Cox7a2) (Fig. 1n) in epididymis adipose tissue (e-WAT) from mice supplemented with L. reuteri ZJ617 compared to mice fed with only HFD. Proteins (UCP1 and ADRB3) (Fig. 1m) and mRNAs (Adrb3, Pgc-1b, Ppara, and Pparg) (Fig. 1n) relevant to the browning process were also upregulated in e-WAT in mice supplemented with L. reuteri ZJ617 compared to mice fed with only HFD, despite a decrease in the mRNA level of Ucp1 gene (Supplementary Fig. 1o). Together, our results indicate that L. reuteri ZJ617 treatment mitigates MetS and may activate the browning process of WAT in HFD-fed mice.

L. reuteri ZJ617 modulates gut microbiota in HFD-fed mice

Previous study has suggested that the improvement of host metabolism by single bacteria or bacterial consortia does not rule out the possibility of microbe-microbe interactions²¹. We thus investigated the impacts of L. reuteri ZJ617 on the gut microbiota by performing 16S ribosomal RNA gene amplicon sequencing on intestinal contents from HFD-fed mice supplemented with or without L. reuteri ZJ617 and LFD-fed mice. The unweighted UniFrac-based principal coordinates analysis (PCoA) revealed a clear clustering of the gut microbiota for each group (Fig. 2a). L. reuteri ZJ617 supplementation resulted in a shift in β-diversity (Fig. 2b) and a reversal of the Firmicutes-to-Bacteroidetes ratio (P = 0.07) (Fig. 2c, d) in comparison to mice fed with only HFD. A random forest classifier was applied to further identify the specific microbiota that was altered by the L. reuteri ZJ617 treatment (P < 0.1). Overall, out of 409 amplicon sequence variants (ASVs), 100 ASVs showed different relative abundances between LFD-fed and HFD-fed mice (Supplementary Fig. 2), and L. reuteri ZJ617 supplementation affected 45 ASVs, with 20 enriched and 25 reduced when compared to HFD-fed mice (Fig. 2e). At the genus level, HFD feeding led to decreased abundances of Parabacteroides, Roseburia, Bacteroides, Muribaculaceae and Parasutterella compared to mice fed an LFD (Supplementary Fig. 2), while L. reuteri ZJ617 supplementation prevented this decline (Fig. 2e). Moreover, L. reuteri ZJ617 supplementation increased the fecal abundance of Lactobacillus at different time points in comparison to mice fed with only HFD (Supplementary Fig. 1p).

**Fig. 2: *L. reuteri* ZJ617 re-programs gut microbiota composition of HFD-fed mice.**

To further understand the functional potential of these reorganized microbial communities, PICRUSt2 was applied to predict metagenome functions. Among the 355 identified pathways, 64 exhibited differential abundance in the intestinal contents of mice supplemented with L. reuteri ZJ617 compared to the HFD-fed mice (P < 0.05). Pathways related to polyamine synthesis were among the top-ranked upregulated pathways (Fig. 3a), and higher levels of the key genes responsible for polyamine production (Fig. 3b) and transportation (Fig. 3c) were detected. Although amplicon-based prediction was unable to distinguish strain-specific functionality due to insufficient resolution, the BlastP analysis (bit score >200) validated the presence of key genes involved in polyamine synthesis in the bacteria genus upregulated by L. reuteri ZJ617 (Fig. 3d, e). This result is consistent with previous studies showing spermidine production capacity in Parabacteroides, Roseburia, and Bacteroide^22,23. Thus, our findings indicate that supplementation of L. reuteri ZJ617 reshapes the composition and function of the microbiota in HFD-fed mice, which may lead to upregulation of key microbial genes involved in polyamine biosynthesis.

**Fig. 3: *L. reuteri* ZJ617 changes microbial function of HFD-fed mice.**

The metabolic benefits of L. reuteri ZJ617 are mediated by the gut microbiota

In order to investigate whether the L. reuteri ZJ617-induced microbial shift directly contributes to metabolic protection, we transplanted fecal microbiota from three experimental groups separately into recipient mice that had been pre-treated with antibiotic mixtures to remove microbiota. The recipient mice were then maintained on HFD for 14 weeks (Fig. 4a). Mice transplanted with HFD microbiota (FHH) showed a marked increase in body weight gain compared to mice transplanted with LFD microbiota (FLH), whereas mice transplanted with HFD + ZJ617 microbiota (FZH) exhibited a significant reduction in weight gain compared to FHH mice (Fig. 4b, c). In addition, compared to FHH mice, FZH mice showed an enhanced ability to clear blood lipids (Supplementary Fig. 3a–c), improved glucose tolerance and insulin sensitivity (Fig. 4d, e), healthier serum AST and ALT levels (Supplementary Fig. 3d, e), and reduced accumulation of adipose depots and adipocyte areas (Fig. 4f–h). There was no significant difference in liver weights or hepatic triglyceride levels between FHH and FZH mice (Supplementary Fig. 3f–h). Notably, FZH mice exhibited similar WAT browning as observed in the donor mice. The mitochondrial density (Fig. 4i and Supplementary Fig 3i) and the expression of “browning” associated marker proteins (ADRB3 and UCP1) (Fig. 4j) and genes (Adrb3, Pgc-1b, Pparg and Cidea) (Fig. 4k) were all upregulated in WAT of FZH mice as compared to FHH mice.

**Fig. 4: Transplantation of *L. reuteri* ZJ617-induced microbiota ameliorates metabolic syndrome in HFD-fed mice.**

We then conducted 16S rRNA sequencing to examine the composition of the intestinal microbiota in the recipient mice. The PCoA analysis revealed a compositional difference between the microbiota of FHH mice and FZH mice (Supplementary Fig. 4a, b). The heatmap showed that 71 ASVs were changed in FZH mice and 90 ASVs were changed in FLH mice (Supplementary Fig. 4c, d). Compared to FHH mice, the abundances of Parabacteroides and Muribaculaceae were significantly higher in FZH mice, but there was no significant difference for Parasutterella, Roseburia, and Bacteroides (Supplementary Fig. 4d). Consistent with HFD-fed mice supplemented with L. reuteri ZJ617, the prediction of metagenome functions indicated that the pathways related to polyamine biosynthesis were more abundant in FZH mice than in FHH mice (Supplementary Fig. 5a). The key genes and transport proteins involved in polyamine production were also enriched in the FZH mice compared to the FHH mice (Supplementary Fig. 5b, c). Together, these results indicate that there exists a causal relationship between microbiota-related polyamine synthesis and the metabolic benefits of L. reuteri ZJ617.

L. reuteri ZJ617 supplementation increases the production of microbiota-derived spermidine

We next performed untargeted metabolomic profiling on intestinal contents to identify potential microbial molecules communicating between the host and the microbiome. A total of 530 metabolites were detected in the intestinal contents. The orthogonal partial least squares-discrimination analysis (OPLS-DA) showed distinct clustering of metabolites among LFD-fed mice, HFD-fed mice, and HFD-fed mice supplemented with L. reuteri ZJ617 (Fig. 5a). Compared to LFD-fed mice, HFD-fed mice exhibited an increase of 52 metabolites and a decrease of 27 metabolites (Supplementary Table 3). L. reuteri ZJ617 supplementation resulted in the upregulation of 8 metabolites and the downregulation of 15 metabolites in HFD-fed mice (Fig. 5b and Supplementary Table 4). The pathway enrichment analysis showed that L. reuteri ZJ617 supplementation had the most significant impact on the arginine and proline metabolism pathway (Fig. 5c), which is involved in polyamine production. In agreement with the prediction of the metagenome functions, spermidine levels were increased after L. reuteri ZJ617 supplementation (Fig. 5b). We then performed targeted polyamine metabolomics on both intestinal contents and serum samples to validate the results. For intestinal contents, most of the metabolites on polyamine metabolism (l-Arginine, Agmatine, S-adenosylmethionine, l-Ornithine, Spermidine) were significantly higher in mice supplemented with L. reuteri ZJ617 than in mice fed with only HFD. Furthermore, supplementation of L. reuteri ZJ617 only increase the concentration of spermidine in serum (Fig. 5d), while the level of spermine was significantly reduced (Fig. 5d). The levels of spermidine were consistently found to be higher in both the intestinal contents and serum of FZH mice than in FHH mice (Supplementary Fig. 6a, b).

**Fig. 5: *L. reuteri* ZJ617 alters the metabolic profile of HFD-fed mice and increases the production of spermidine.**

Previous research has established that spermidine is obtained orally through dietary sources, such as the polyamine precursor arginine, or through the synthesis by commensal bacteria²⁴. Whether spermidine was produced by L. reuteri ZJ617 or by commensal gut bacteria remains unknown in our setting. The genomic information revealed that L. reuteri ZJ617 only expresses genes that encode spermidine/putrescine transport proteins (Supplementary Table 5), but not genes that are essential for spermidine synthesis. Therefore, we detected a decrease of spermidine in the supernatant of cultured L. reuteri ZJ617 (Supplementary Fig. 6d). Notably, we found that metK gene, that encode S-adenosylmethionine synthetase, is expressed by L. reuteri ZJ617 (Supplementary Table 5). This aligns with the observation of increased S-adenosylmethionine (SAM) levels in the intestinal contents of mice supplemented with L. reuteri ZJ617 (Fig. 5d), as well as in the intestinal contents of FZH mice (Supplementary Fig. 6c). The in vitro cultivation of L. reuteri ZJ617 revealed additional evidence supporting the production of SAM by L. reuteri ZJ617 (Fig. 5e). Because SAM is the precursor of spermidine, our findings suggest that L. reuteri ZJ617 is able to provide substrates for other spermidine-producing bacteria.

To better understand the efficacy of L. reuteri ZJ617 in facilitating the production of microbiota-derived spermidine, we applied a gastrointestinal tract simulation system to examine the SAM levels in the simulated gut lumen that was inoculated with fecal microbiota from individuals with obesity (Fig. 5f). Consistent with the results obtained from mice metabolomics (Fig. 5d), SAM levels were observed to be higher if L. reuteri ZJ617 was co-cultured in the simulated gut lumen (Fig. 5g). As the same time, both the abundance of spermidine-producing bacteria and the level of spermidine increased in the simulated gut lumen co-cultured with L. reuteri ZJ617 in vitro (Supplementary Fig. 6e–g and Fig. 5h). Importantly, the addition of SAM to the simulated gut lumen also increases the abundance of spermidine-producing bacteria and the production of spermidine (Supplementary Fig. 6e–g and Fig. 5h). Although the BlastP analysis (Fig. 3d) and previous studies have demonstrated the spermidine production capacity of Parabacteroides, Roseburia, and Bacteroides^22,23, in this study, Bacteroides was reconfirmed to have ability to convert SAM into spermidine in in vitro cultivation. Spermidine level in the supernatant significantly increased after co-culturing Bacteroides with SAM, compared with the supernatant containing only SAM. (Supplementary Fig. 6h). These findings collectively suggest that L. reuteri ZJ617 promotes spermidine production in fecal bacteria from individuals with obesity.

Microbiota-derived spermidine ameliorates metabolic syndrome and promotes the browning of WAT

To further assess the importance of spermidine biosynthesis in mediating the metabolic benefits of L. reuteri ZJ617, we added spermidine directly into the drinking water of mice given an HFD (Fig. 6a). Compared to mice fed with only HFD, spermidine supplementation markedly lowered body weights (Fig. 6b, c), improved hyperlipidemia and insulin tolerance (Fig. 6d, e, h), reduced liver weights and triglycerides (Fig. 6i, j), and alleviated hepatic steatosis (Fig. 6k), although did not improve LDL-C levels and glucose tolerance (Fig. 6f, g). Importantly, spermidine supplementation also promoted energy metabolism (Supplementary Fig. 7a–c), elevated WAT temperature (Fig. 7a, b), reduced WAT accumulation and adipocyte areas (Fig. 7c–e), augmented mitochondria density (Fig. 7f and Supplementary Fig. 7d), and upregulated the expression of mitochondrial biogenesis genes (Cox5b, Cox7a1, Cox7a2) (Fig. 7h), browning-related proteins (ADRB3 and UCP1) (Fig. 7g) and mRNAs (Adrb3, Ppara, and Pparg) (Fig. 7h).

**Fig. 6: Microbial production of spermidine ameliorates MetS in HFD-fed mice.**

**Fig. 7: Microbiota-derived spermidine promotes browning of mouse white adipose tissue and 3t3-L1 adipocytes.**

As a comparison, we administered a polyamine biosynthesis pathway inhibitor, diminazene aceturate (DA) to mice via gavage. DA has been found to hinder the formation of S-adenosylmethionine^25,26, and block the activity of spermidine/spermine N1-acetyltransferase, hence inhibiting the back conversion of spermidine and spermine into putrescine^27,28. Therefore, DA could cut off both putrescine and S-adenosylmethionine-derived polyamine synthesis pathways. We found that DA administration through intraperitoneal injection or oral gavage had no effect on spermidine levels in WAT of mice fed with either a normal diet or HFD (Supplementary Fig. 7e, f), but it significantly decreased spermidine level in intestinal contents from HFD-fed mice (Supplementary Fig. 7g). These results indicate that the dosage of DA we used can reduce spermidine synthesis by the gut microbiota while having a minor impact on spermidine production in adipose tissue. Remarkably, when DA was administered to HFD-fed mice supplemented with L. reuteri ZJ617, the metabolic benefits of L. reuteri ZJ617 were largely abolished (Fig. 6b–k), and the non-shivering thermogenesis in WAT was also decreased (Fig. 7a–h).

Since spermidine reaches target tissues through rapid plasma turnover to exert physiological functions²⁹, we measured spermidine concentrations in WAT from HFD-fed mice that underwent different treatments. As compared to control mice given only an HFD, oral administration of spermidine significantly increased spermidine levels in WAT, with an increasing trend observed for L. reuteri ZJ617 (P = 0.08). However, there was no significant difference for HFD-fed mice given L. reuteri ZJ617 in combination with DA (Fig. 7i). Similarly, FZH mice showed an increased spermidine level in WAT as compared to FHH mice (Supplementary Fig. 7h).

In order to further confirm a direct involvement of spermidine in adipocyte browning, we treated the preadipocyte cell line 3T3-L1 with spermidine during its chemical-induced differentiation. The addition of spermidine resulted in a significant reduction in lipid droplets (Fig. 7j and Supplementary Fig. 7i) and an increase in the levels of adipocyte browning-related proteins (ADRB3 and UCP1) (Fig. 7k). Together, our data provide mechanistic evidence that spermidine acts as a potential molecule that mediates the metabolic benefits of the L. reuteri ZJ617 in improving MetS and promoting adipocyte browning.

Discussion

There is strong evidence from randomized clinical trials and meta-analyses that the administration of L. reuteri is associated with a lower risk of MetS^10,11,12,30. In this study, we showed that intake of L. reuteri ZJ617 improves MetS in HFD-induced obese mice. We found no clear adverse effects in mice following long-term L. reuteri ZJ617 exposure, as we had seen in our prior study in pig and mouse models fed a normal diet^18,31. We discovered that L. reuteri ZJ617 impacts the composition and function of the gut microbiome while offering metabolic benefits to the host. L. reuteri ZJ617 modulates energy metabolism, promotes WAT browning, and mitigates MetS symptoms by providing substrates for the microbiota to synthesize microbial-derived spermidine.

Although multiple studies have reported L. reuteri supplementation can modulate gut microbiota of mouse models and humans with metabolic disorders^11,32,33, few studies have focused on the modulation of host metabolism by specific microbial metabolites derived from L. reuteri or L. reuteri-modulated commensal bacteria. In this study, we discovered that microbial-derived spermidine is the major contributor to the metabolic benefits of L. reuteri ZJ617, and that L. reuteri ZJ617 can provide a substrate for commensal bacteria to synthesize spermidine. Because the metK gene has been identified in various Lactobacillus species in another study³⁴, our study indicates that spermidine synthesis via reciprocity may be a key reason for the probiotic effects of numerous Lactobacilli.

A growing body of literature supports the role of spermidine in modulating host metabolism. One study has found that daily intake of spermidine was negatively correlated with body mass index (BMI), waist circumference, and HOMA-IR index in humans³⁵. The mechanism by which spermidine exerts its metabolic benefits including gut barrier enhancement³⁵, hepatic mitochondrial function improvement³⁶, BAT activation, and muscle adaptation³⁷. However, few studies have shown that spermidine impacts on WAT. Since excess WAT accumulation is a major risk factor for developing MetS³⁸, activating WAT browning is critical for improving human health. Herein, we found that both L. reuteri ZJ617 and spermidine improve whole-body energy metabolism and promote WAT browning. A previous study has confirmed that spermidine promotes WAT lipolysis in an ADRBs-dependent way³⁹. Spermidine mimics the β-adrenergic stimulation activating ADRBs receptor, thereby increasing cyclic AMP levels, and driving protein kinase (PKA) signaling to promote lipolysis³⁹. These released fatty acids via adipocyte lipolysis subsequently promote the activation of transcriptional regulators that drive mitochondrial biogenesis and the expression of thermogenic genes³. The most well-studied thermogenic effector is the UCP1 protein. Even though we observed an upregulated expression of the Ucp1 gene and downregulated expression in UCP1 protein in HFD mice, this is not unusual as comparable findings have been reported in earlier studies⁴⁰. This opposite phenomenon may be attributed to the complex regulation of gene transcription and protein translation⁴¹. Our findings suggest that WAT browning may serve as a mechanism through which L. reuteri ZJ617 or spermidine alleviates MetS. However, given the baseline expression of UCP1 in HFD mice is barely detectable, we acknowledged that the mild browning induced by L. reuteri ZJ617 may not completely account for leanness and an improved metabolic phenotype. Therefore, the specific contribution of UCP1 and ADRB3 proteins to the browning process induced by L. reuteri ZJ617 and the spermidine remains to be elucidated in further studies. Furthermore, spermidine has been shown to exert a range of beneficial effects on cardiovascular disease protection, lifespan extension⁴², bone loss prevention⁴³, antitumor immunity⁴⁴, and oocyte quality⁴⁵ improvement. Given these diverse biological roles, the increased spermidine production induced by L. reuteri ZJ617 may have broad physiological implications that go well beyond metabolic diseases.

Our study has proven the significance of SAM in mediating microbial spermidine synthesis and the metabolic benefits of Lactobacillus. Further studies are needed to generate high-yield SAM-producing Lactobacillus strains. Integrating synthetic biology and metabolomics, as previously done to optimize spermidine production in engineered probiotics⁴⁶, could be key to this advancement. These methodologies provide promising strategies for developing probiotic strains with targeted metabolic outputs, facilitating the translation from laboratory-scale research to semi-industrial-scale applications. Additionally, because L. reuteri ZJ617 has a regulatory effect on promoting spermidine production in gut microbes from individuals with obesity, clinical experiments are needed to better assess the effects of long-term use of L. reuteri ZJ617 on managing metabolic-related diseases.

In summary, our work revealed L. reuteri ZJ617 mitigates MetS via promoting spermidine synthesis in the intestine by supplying substrate to spermidine-producing bacteria and preventing those bacteria from declining (Supplementary Fig. 8). Spermidine produced by L. reuteri ZJ617 improves MetS are linked to enhance whole-body energy metabolism, activate WAT thermogenesis, and improve insulin resistance. Our work may serve as a foundation for the development of a microbiota-based therapeutic approach to treat MetS in clinics.

Methods

Ethics declarations

All animal experiments were performed in accordance with the “Regulation for the Use of Experimental Animals” of Zhejiang Province, China, and approved by the Animal Care and Use Committee of Zhejiang University (Ethics Code Permit Number: ZJU20170529; ZJU20240770). Fecal samples from healthy volunteers with obesity (BMI ≥28) was performed with the approval of the Ethical Committee of The Second Affiliated Hospital of Zhejiang University (Ethics Code Permit Number: 2024-1195). All participants provided written informed consent for sample collection and subsequent experiments.

Animals and L. reuteri ZJ617 administration

Four-week-old male, specific pathogen-free (SPF) C57BL/6J mice were obtained from Shanghai SLAC Laboratory Animal Co., Ltd. (Shanghai, China). Before the onset of the experiment, they were acclimatized to the environment (23 ± 1 °C, 12-h light-dark cycle, 50% ± 5 humidity) with free and unlimited access to food and drinking water for one week. Then mice were randomly assigned into three groups (3 cages/group): LFD (Low-fat diet, containing 70% kcal from carbohydrate and 10% kcal from fat (~5.5% kcal from soybean Oil and ~4.5% from kcal Lard); PD450J), HFD (High-fat diet, containing 20% kcal from carbohydrate and 60% kcal from fat (~5.5% kcal from soybean Oil and ~54.5% kcal from Lard); PD6001) and HFD supplement with L. reuteri ZJ617. All diets were from Changzhou SYSE Bio-Tec. Co., Ltd. (Changzhou, China), and the diet composition is given in Supplementary Table 1. The mice were allowed free access to food and were maintained for 14 weeks.

L. reuteri ZJ617 had previously been isolated from piglet small intestines and stored in 20% glycerol at −80 °C until usage. The strain was anaerobically cultured for 18 h in sterilized De Man Rogosa and Sharpe (MRS) medium at 37 °C. Cultures were collected in the log phase and diluted to 10⁹ colony-forming units/mL in drinking water. There was no significant difference in the number of alive L. reuteri ZJ617, after placement in water for 24 h. The drinking water was refreshed every day. The abundance of Lactobacillus was calculated using plate colony-counting.

Fecal microbiota transplantation

The procedure of fecal microbiota transplantation was done according to a previous study and modified⁴⁷. Feces were collected from the donor LFD, HFD, and HFD + ZJ617 groups, respectively, at the end of week 14, and the pooled sample in each group was used for the following experiment. The fecal pellets were diluted with sterile PBS (100 mg/mL). The mixtures were centrifuged at 1000×g, 4 °C for 5 min. The supernatant was transferred into a new tube and centrifuged for 5 min at 15,000×g to precipitate the bacteria. Then, the bacterial pellets were resuspended in 600 μL sterile PBS, and an equal volume of 40% sterile glycerol was added. The resuspended bacteria were stored at -80 °C for later use with transplantation.

Four-week-old male C57BL/6J mice were acclimatized for 1 week, then their intestinal microbiota was first depleted by an antibiotic mixture ((vancomycin (0.5 g/L), neomycin sulfate (1 g/L), metronidazole (1 g/L), and ampicillin (1 g/L)) in drinking water for 1 week. Then, the 100 μL of bacteria suspension was transplanted to the mice that had been pre-treated with antibiotics via oral gavage for 14 weeks (three times per week). All mice were fed the same diet, HFD, for 14 weeks.

Histological analysis

After euthanasia, the liver and adipose tissues of mice were preserved in 4% paraformaldehyde (4% PFA). The fixed paraffin-embedded sample sections were stained with hematoxylin and eosin (H&E) to assess liver steatosis and adipocyte size, respectively, followed by microscopical examination. All processes were performed according to the manufacturer’s instructions. Adipocyte sizes were quantified by Cellpose 2.0 v and Image J 1.52 v. The slices were collected and evaluated in a blinded manner.

Transmission electron microscope

Adipose tissues were preserved in 2.5% glutaraldehyde for more than 4 h and then were post-fixed with 1% OsO₄. The double-fixed Spurr resin-embedded sample sections were stained by uranyl acetate and alkaline lead citrate for 5 min, respectively, and then mitochondria were observed with a TEM (Hitachi Model H-7650). All processes were performed according to the instructions of the Bio-ultrastructure analysis Laboratory of Analysis center of Agrobiology and environmental sciences, Zhejiang University. The slices were collected and evaluated in a blinded manner.

Glucose homeostasis

For oral glucose tolerance testing (OGTT), all mice were placed in clean cages and provided with water but without food. After 6 h of fasting, the mice were administrated an oral glucose (2 g/kg, Sigma-Aldrich, 158968). Blood glucose levels were measured before and after oral glucose administration at 15, 30, 60, 90, and 120 min and determined with a glucose meter (Accu-Chek Performa, Roche) on blood samples collected from the tip of the tail vein during week 12.

For insulin tolerance testing (ITT), all mice were placed in clean cages and provided with water but no food. After five hours of fasting, the mice were injected with insulin (0.75 U/kg, Sigma-Aldrich, I2643), and blood glucose levels were measured before and after injection at 15, 30, 60, 90, and 120 min after injection during week 13.

Biochemical analysis

Blood samples were centrifuged at 3000 × g for 15 min at 4 °C to produce serum samples. The levels of serum AST, ALT, TG, TC, and LDL-C were determined using the corresponding assay kits (C010-2-1, C009-2-1, A110-1-1, A111-1-1, and A113-1-1, respectively; Nanjing Jiancheng Bioengineering Institute, Nanjing, China). Serum insulin levels were measured using ELISA kits (H203-1-1, Nanjing Jiancheng Bioengineering Institute, Nanjing, China). Liver tissues were homogenized with ethanol and centrifugated at 2500×g for 10 min at 4 °C, and the supernatant was used to determine TG. All indexes were used a multifunctional microplate reader (TECAN, Switzerland), according to the manufacturer’s protocols.

Real-time qPCR

Total RNA was extracted using a SteadyPure Universal RNA Extraction kit (Accurate Biotechnology, AG21017, China) according to the manufacturer’s protocols. RNA concentrations were equalized and converted to cDNA using a kit (Accurate Biotechnology, AG11728, China). Gene expression was measured by qPCR detection (Bio-Rad, CFX96, USA) systems using SYBR Green (Accurate Biotechnology, AG11701, China). Expression was normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) control. The primer is given in Supplementary Table 2.

Western blot

White adipose tissue and cells were lysed with RIPA buffer containing PMSF, proteinase inhibitor, and phosphatase inhibitors (Beyotime Technology, Shanghai, China) in an ice bath followed by centrifugation at ~13,000 × g for 10 min. The supernatant was collected and denatured in a sample buffer and resolved with SDS-PAGE. Samples were transferred to PVDF membranes with a pore size of 0.45 μM. Individual proteins were detected using 1:1000 dilution of primary antibodies, as follows: ADRB3 (1:1000, Abcam, ab94506, polyclonal antibody), UCP1 (1:1000, Abcam, ab234430, polyclonal antibody) and β-actin (1:1000, Cell Signaling Technology, 4970, polyclonal antibody). Proteins were visualized on film with horseradish peroxidase–conjugated secondary antibodies diluted 1:5000.

16S rDNA amplicon sequencing

The V3-V4 region of the bacteria 16S ribosomal RNA genes were amplified using primers 341 F 5′-CCTACGGGRSGCAGCAG)-3′ and 806 R 5′-GGACTACVVGGGTATCTAATC-3′. PCRs were performed, and the library was constructed using Qubit. The purified amplicons were pooled in equimolar amounts, and paired-end sequenced on an Illumina MiSeq PE250 platform (Study1)/NovaSeq PE250 platform (Study2) (Illumina, San Diego, USA) according to the manufacturer’s protocols. ASVs were selected via standard clustering with 100% similarity using Deblur. Each representative tag was assigned to a taxon using the RDP Classifier. The QIIME2 was implemented for ASVs profiling.

PICRUSt2 for prediction of metagenome functions

PICRUSt2 was utilized to predict metagenome functions⁴⁸. Gene banks such as Kyoto Encyclopedia of Genes and Genomes (KEGG Orthologous, KO), Enzyme Commission numbers (EC no.), and Clusters of Orthologous Groups (COG) were used to support functional gene and pathway profiles. All predicted pathways and differently expressed genes were obtained using a Wilcoxon test.

Metabolomic profiling of intestinal contents

For metabolomic analysis, intestinal contents were collected and immediately snap-frozen in liquid nitrogen. The metabolites were analyzed by gas chromatography time-of-flight mass spectrometer (GC-TOF-MS), according to our previous study⁴⁹. The pre-weighted intestinal contents were extracted and homogenized with a pre-cold mixture (methanol/chloroform, v: v = 3:1). The centrifuged and speed-vacuum-dried metabolites were solubilized in 40 μL of methoxyamination hydrochloride (20 mg/mL in pyridine) and then incubated at 80 °C for 30 min, then derivatized by 60 μL of BSTFA regent (1% TMCS, v/v) at 80 °C for 1.5 h. The cooled samples were mixed with fatty acid methyl esters (FAMEs; 5 μL in chloroform) and injected into the GC-TOF-MS system for detection and analysis.

For the data of the metabolites of intestinal contents, the abundance differences were obtained using a Wilcoxon test. OPLS-DA was conducted to identify the discrimination of variables. Differential metabolites were defined as those with variable importance in the projection (VIP) >1.0 obtained from OPLS-DA and P < 0.05.

In vitro gastrointestinal tract simulation

In vitro fecal microbiota fermentation was conducted with a gastrointestinal tract simulation system as described previously with some modification^50,51. The feces were collected from six adult volunteers with obesity (BMI ≥28, aged 20–25) who had not taken any antibiotics and probiotics in the past 6 months. The fecal slurry (1:9; w/v) was prepared by diluting the homogenized fecal sample in sterile PBS medium, which was added into the colon simulation bioreactor with nutrient medium (1:9; v/v). After 24 h of inoculation and culture, fresh nutrient medium was added into the bioreactor and waste was drained at a certain rate (Stage 1) to maintain the normal growth of microorganisms. L. reuteri ZJ617 (10⁹ CFU) and S-adenosylmethionine (0.05% w/v; equal to 0.8 g/day for an adult according to previous study⁵²) was added to the reactor. The fermentation lasted for 6 days (Stage 2). During the entire fermentation process, the culture was automatically supplemented with 0.5 mol/L NaOH solution and diluted with HCl to adjust the pH to 6.8. The speed of the stirring rotor was set at 80 r/min. The temperature was kept at 37 °C modulated by the heating and cooling system. Anaerobic conditions were generated by flushing the headspace of all reactions and medium vessels with N₂ for 30 min and three times per day. Each sample was collected from three independent fermentation waste extraction at the beginning and end of stage 2 for spermidine-producing bacteria or spermidine determination.

Bacteroides uniformis

Bacteroides uniformis were obtained from our previous study⁵³. Briefly, the experiment consisted of three groups: medium only (Control), medium supplemented with 10 μM S-adenosylmethionine (SAM), and medium supplemented with SAM and B. uniformis (B. uniformis + SAM). All groups were placed in a vinyl anaerobic chamber (Coy Drive 14,500, USA) and anaerobically cultured at 37 °C for 48 h. At the end of the experiment, all samples were harvested for spermidine determination.

Concentration of spermidine

The concentration of spermidine in intestinal contents, serum, adipose tissue, and bacteria fermentation supernatant was analyzed by UHPLC-MS/MS or HPLC, as previously described and modified⁵⁴. The weighted intestinal contents, serum and adipose tissues and the bacteria fermentation supernatant were extracted and homogenized with ice-cold perchloric acid solution (0.4 mol/L), the bacteria were disrupted by sonication. All samples were derivatized after pH was adjusted to 9.0. Dansyl chloride acetone solution (10 mg/mL, Aladdin, D133513) was applied for derivatization. Nitrogen-dried metabolites were re-solubilized in HPLC-grade acetonitrile (Sigma-Aldrich, 34851) and injected into the HPLC system while obtaining spermidine ion traces.

Spermidine supplementation and inhibitor treatment

Four-week-old male C57BL/6J mice were acclimatized for one week. HFD-fed mice were supplemented with spermidine (20 mg/kg) in drinking water for 14 weeks, The dosage was determined based on a previous study³⁵. The drinking water was refreshed every day. The concentration of spermidine in drinking water was adjusted as w/v based on the changes in body weight and water consumption. For the spermidine synthesis inhibitor, HFD-fed mice were supplemented with L. reuteri ZJ617 and oral gavage with diminazene aceturate (2.5 mg/kg) daily for 14 weeks. The mice were allowed free access to food and were maintained for 14 weeks.

Metabolic rate measurements

The mouse metabolic rate was assessed after 4 weeks of treatment with L.reuteri ZJ617 or 14 weeks of treatment with spermidine, using the TSE PhenoMaster System (TSE PhenoMaster, Germany) following the manufacturer’s instructions. Before the metabolic rate was monitored, mice were individually caged for 24 h to acclimate to the system.

Cell culture and staining

The 3T3-L1 preadipocytes (SCSP-5038) were purchased from the National Collection of Authenticated Cell Cultures, grown in high-glucose DMEM (Gibco) supplemented with 10% calf serum (Sigma-Aldrich, B7447) and 1% penicillin-streptomycin at 37 °C in an incubator containing 5% CO₂, followed by feeding with fresh medium every 2 days to reach confluence. For beige adipocyte differentiation, the cells were induced with an induction medium containing DMEM, 10% FBS, 0.5 mM isobutylmethylxanthine, 1 μM dexamethasone, 4.5 μg/mL insulin, and 1 μM rosiglitazone with or without spermidine or PBS for 48 h, and further growth medium was supplemented with insulin with or without spermidine or PBS every 2 days for 6 days. The cultured 3T3-L1 adipocytes were stained using an Oil Red O staining Kit (Solarbio, G1262, China) according to the manufacturer’s protocols. The images of the ORO staining were recorded with a microscope and quantified by Image J 1.52v. The images were collected and evaluated in a blinded manner.

Statistics and reproducibility

For assessing differences between the two groups, a two-tailed t-test was performed. For differences among more than two groups, a one-way analysis variance (ANOVA) was performed, followed by Dunnett tests (Study1 and Study2) or Šídák tests (Study3). All data are shown as means ± SD. Statistical analyses were performed using GraphPad Prism 9.0v (USA). P < 0.05 was considered statistically significant. Results from representative experiments (such as micrographs) were obtained from at least three independent fields of view with similar results.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Data availability

The sequence data from all 16S rRNA sequencing experiments have been deposited in the National Center for Biotechnology Information Sequence Read Archive (SRA) under accession number PRJNA945511. For the metabolome data have been deposited in the OMIX, China National Center for Bioinformation, under accession number OMIX008190. The genome sequencing and assembly of Lactobacillus reuteri ZJ617 have been deposited in the Genome Warehouse in the National Genomics Data Center under accession number PRJCA033409. All data supporting the findings of this study are available in the manuscript or Supplementary Data. Source data are provided with this paper.

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Acknowledgements

We thank Ms. Junhong Wang and Dr. Weiren Dong from the lab platform of the College of Animal Sciences, Zhejiang University, for their excellent technical guidance and assistance. We specially thank Dr. Huiqing Yu and professor Guangxu Meng from Institute of Immunity and Infection, Chinese Academy of Sciences for kindly providing the TSE PhenoMaster System and technical support, and professor Xuan Zhu from School of Food Science and Biotechnology, Zhejiang Gongshang University for kindly providing the Gastrointestinal Tract Simulation System. This study was supported by grants from the National Key R&D Program of China (2022YFD1300602), the Key R&D Projects of Zhejiang Province (2023C02026), both awarded by H.W.

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The Key Laboratory of Molecular Animal Nutrition, Ministry of Education, College of Animal Sciences, Zhejiang University, Hangzhou, Zhejiang, 310058, PR China
Yanfei Ma, Yifan Zhong, Wenjie Tang, Teresa G. Valencak, Jingliang Liu, Zhaoxi Deng, Jiangdi Mao, Shanshan Wang & Haifeng Wang
The Second Affiliated Hospital of Zhejiang University, Hangzhou, Zhejiang, 310009, PR China
Daren Liu
Institute of Translational Medicine, Zhejiang University School of Medicine, Hangzhou, Zhejiang, 310029, PR China
Yuhao Wang

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Yanfei Ma
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Y.M. and H.W. planned the project and the experiments. Y.M., H.W., Y.W., and Y.Z. analyzed the data; Y.M., W.T., J.L., Z.D., J.M., D.L., and S.W. performed the experiments; Y.M. and H.W. wrote the original draft; Y.M., Y.W., H.W., and T.G.V. were responsible for writing review and editing. Funding acquisition was the responsibility of H.W.

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Ma, Y., Zhong, Y., Tang, W. et al. Lactobacillus reuteri ZJ617 attenuates metabolic syndrome via microbiota-derived spermidine. Nat Commun 16, 877 (2025). https://doi.org/10.1038/s41467-025-56105-4

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Received: 27 March 2023
Accepted: 09 January 2025
Published: 21 January 2025
DOI: https://doi.org/10.1038/s41467-025-56105-4

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Abstract

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Introduction

Results

L. reuteri ZJ617 alleviates the symptoms of metabolic syndrome in HFD-fed mice

L. reuteri ZJ617 modulates gut microbiota in HFD-fed mice

The metabolic benefits of L. reuteri ZJ617 are mediated by the gut microbiota

L. reuteri ZJ617 supplementation increases the production of microbiota-derived spermidine

Microbiota-derived spermidine ameliorates metabolic syndrome and promotes the browning of WAT

Discussion

Methods

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Animals and L. reuteri ZJ617 administration

Fecal microbiota transplantation

Histological analysis

Transmission electron microscope

Glucose homeostasis

Biochemical analysis

Real-time qPCR

Western blot

16S rDNA amplicon sequencing

PICRUSt2 for prediction of metagenome functions

Metabolomic profiling of intestinal contents

In vitro gastrointestinal tract simulation

Bacteroides uniformis

Concentration of spermidine

Spermidine supplementation and inhibitor treatment

Metabolic rate measurements

Cell culture and staining

Statistics and reproducibility

Reporting summary

Data availability

References

Acknowledgements

Author information

Authors and Affiliations

Contributions

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Competing interests

Peer review

Peer review information

Additional information

Supplementary information

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