Dietary PUFA-derived gut microbial metabolites

Microbes transform PUFAs into a variety of FAs, such as LA-derived metabolites (Supplementary Fig. 1a)17 and αLA-derived metabolites (Supplementary Fig. 1b)18. We first examined the gut-microbial PUFA-metabolite profiles in the cecum of normal chow (NC)-fed and HFD-fed mice, finding that LA-derived metabolites existed at predominantly higher levels as compared with those associated with αLA-derived metabolites (Supplementary Fig. 1c), with 10-hydroxy-cis-12-octadecenoic acid (HYA), 10-hydroxyoctadecanoic acid (HYB), 10-hydoroxy-trans-11-octadecenoic acid (HYC), 10-oxo-cis-12-octadecenoic acid (KetoA), 10-oxo-octadecanoic acid (KetoB), and 10-oxo-trans-11-octadecenoic acid (KetoC) in LA-derived metabolites specifically attenuated by HFD-feeding for 2 weeks (Fig. 1a). Moreover, by comparing these LA-derived metabolites (HYA, HYB, HYC, KetoA, KetoB, and KetoC) in the cecum of NC- and HFD-fed mice, we observed a significantly reduced level of these metabolites in mice fed a HFD for 2 weeks (Fig. 1b). However, HFD supplemented with LA increased cecal levels of HYA (the initial gut-microbial PUFA-metabolite derived from LA) and KetoA, whereas levels of all metabolites were markedly decreased in both NC- and HFD-fed germ-free (GF) mice (Fig. 1b). Moreover, 16 S rRNA gene amplicon sequencing confirmed that HFD supplemented with LA altered the relative abundance of the major phyla constituting the gut microbiota (Fig. 1c). Specifically, in agreement with previous studies6, the abundance of Firmicutes markedly increased and that of Bacteroidetes markedly decreased in HFD-fed mice; however, the abundance of Firmicutes was decreased by LA supplementation (Fig. 1c). Additionally, we confirmed that HFD feeding altered gut microbiota composition, as indicated by principal coordinate analysis (PCoA) based on taxonomic datasets (Fig. 1d). Moreover, hierarchical clustering of individual families confirmed the effect of HFD and LA-supplemented HFD on the gut microbiome (Fig. 1e). Although HFD feeding was associated with a drastically decreased abundance of the Lactobacillaceae family belonging to Firmicutes, interestingly, LA supplementation contributed to an increase in the abundance of Lactobacillaceae (Fig. 1e) and a significant expansion of the Lactobacillus genus (Fig. 2a). Moreover, we examined what species were related to HYA production in the Lactobacillus genus by in vitro bacterial-culture screening. Lactobacillus salivarius and Lactobacillus gasseri efficiently produced HYA from LA in 22 Lactobacillus strains, whereas Lactobacillus acidophilus and Lactobacillus johnsonii produced very little HYA (Supplementary Table 1). Similarly, L. salivarius and L. gasseri, as HYA-producing bacteria, were markedly decreased in HFD-fed mice and significantly increased following LA supplementation, whereas L. acidophilus and L. johnsonii, as HYA non-producing bacteria, did not change following LA supplementation (Fig. 2b). Furthermore, we observed suppressed expression of Cla-hy, a key LA-metabolizing enzyme characterized in gut microbes (Supplementary Fig. 1a), in HFD-fed mice and that this suppression was alleviated by LA supplementation, whereas levels of Cla-dh and Cla-er (Supplementary Fig. 1a) did not change following LA supplementation (Fig. 2c). These findings collectively demonstrated that HFD feeding altered gut microbial composition and inhibited the production of PUFA metabolites by the gut microbes; however, PUFA supplementation restored the abundance of the Lactobacillus and the gut microbial PUFA-metabolite HYA.

Fig. 1 Effects of dietary PUFAs on gut microbiota composition and PUFA metabolites. a Heat map of gut microbial PUFA metabolites in cecal content (n = 8 per group). b LA-derived gut microbial PUFA metabolites in cecal content were quantified (n = 7, 10, 8, 8, and 8 per group for HYA; n = 8,10, 8, 8, and 8 per group for HYB; n = 7, 10, 8, 8, and 10 per group for HYC; n = 8, 10, 8, 8, and 10 per group for KetoA; n = 7, 10, 7, 8, and 8 per group for KetoB; n = 8, 9, 8, 8, and 8 per group for KetoC). *P < 0.05 (Tukey–Kramer test). c–e Gut microbial composition was evaluated in order to determine the relative abundance of microbial taxa (c), diversity (d), and abundance of the bacterial domain at the family level (e) (n = 8, 10, and 7 per group). (−)_NC represents normal chow-fed mice, and (−)_HFD represents high-fat diet-fed mice. q < 0.05. Results are presented as means ± SE. Source data are provided as a Source Data file 1 Full size image

Fig. 2 HYA-producing lactic acid bacteria. a The abundance of Lactobacillus was analyzed by qPCR (n = 9, 8, and 9 per group). b The abundance of the Lactobacillus strains (n = 7, 8, and 7 per group) and c the relative mRNA expression of metabolite-synthesizing enzymes (Cla-hy, Cla-dh, and Cla-er; see Supplementary Fig. 1) (n = 7 per group for Cla-hy; n = 8 per group for Cla-dh and Cla-er) in cecum were analyzed by qPCR and qRT-PCR. **P < 0.01, *P < 0.05 vs. NC (Tukey–Kramer test). ##P < 0.01; #P < 0.05 vs. (−) (Tukey–Kramer test). (−)_HFD represents high-fat diet-fed mice. Results are presented as means ± SE. Source data are provided as a Source Data file 2 Full size image

Microbial PUFA metabolites improve host metabolic condition

We then examined the effects of gut-microbial PUFA metabolites on host energy regulation in a mouse model of HFD-induced obesity. HYA is an initial product of gut-microbial metabolites from dietary LA (Supplementary Fig. 1a). In this experiment, 4-week-old mice were fed HFD (control) or HFD supplemented with either 1% HYA as a gut-microbial metabolite or 1% LA, a precursor of HYA for 12 weeks (Supplementary Table 2). We found that the body weight of HYA-fed mice was significantly lower as compared with that of control and LA-fed mice during growth (Fig. 3a). In addition, the fat mass of white adipose tissue (WAT) was significantly lower in HYA-fed mice than in control and LA-fed mice at 16 weeks of age (Fig. 3b). Moreover, we found a significant decrease in WAT adipocyte size in HYA-fed mice (Fig. 3c), and the food intake of HYA- and LA-fed mice was significantly lower than that of control mice (Fig. 3d). Furthermore, HFD-induced insulin resistance and impaired glucose tolerance, as determined by the insulin tolerance test (ITT) and glucose tolerance test (GTT), respectively, were significantly attenuated in HYA-fed mice as compared with control and LA-fed mice (Fig. 3e, f). Moreover, the plasma glucose and total cholesterol levels of HYA-fed mice were significantly lower than those of control mice (Fig. 3g, h), whereas triglyceride levels were similar among control, HYA-, and LA-fed mice (Fig. 3i). Additionally, we found that levels of the plasma peptide YY (PYY) were significantly higher in HYA-fed mice than in control mice (Fig. 3j), and similarly, that plasma GLP-1 levels were markedly higher in HYA-fed mice than in control and LA-fed mice (Fig. 3k), whereas the plasma insulin levels of HYA-fed mice were significantly lower than those of control mice (Fig. 3l). Furthermore, we observed a significant increase in the level of fecal triglycerides of HYA-fed mice (Fig. 3m). These findings suggested that HYA supplementation suppressed appetite and improved metabolic condition, thereby inducing greater resistance to HFD-induced obesity, even in the presence of PUFAs. In addition, we performed the same experiment under 0.5% HYA-supplemented HFD-fed conditions equivalent to physiologically relevant concentrations of HYA in the cecum of NC-fed mice (Supplementary Fig. 2a). The results showed that 0.5% HYA supplementation suppressed appetite and improved metabolic condition, thereby inducing greater resistance to HFD-induced obesity, similar to 1% HYA supplementation (Supplementary Fig. 2b–m).

Fig. 3 Gut microbial PUFA metabolites improve host metabolic conditions. Changes in a body weight and b representative macroscopic appearance and tissue weights (n = 14 per group). Scale bar; 1 cm. epi, epididymal; peri, perirenal; sub, subcutaneous. c Hematoxylin–eosin (H&E)–stained WAT and the mean area of adipocytes (n = 8 per group). Scale bar, 400 μm. d Daily food intake measured at 7 weeks of age (n = 5 per group). e ITT (n = 10 per group) and f GTT (n = 10 per group) were analyzed at 13–14 weeks of age. g Blood glucose (n = 14 per group), h total plasma cholesterol (n = 10, 9, and 10 per group), i triglyceride (n = 10 per group), j PYY (n = 10, 9, and 8 per group), k GLP-1 (n = 8, 9, and 9 per group), and l insulin (n = 7, 8, and 8 per group) levels were measured at the end of the experimental period. m Fecal triglyceride levels were measured at 16 weeks of age (n = 10 per group). **P < 0.01; *P < 0.05 vs. control (Tukey–Kramer test). ##P < 0.01; #P < 0.05 vs. HYA (Tukey–Kramer test). Results are presented as means ± SE. Source data are provided as a Source Data file 3 Full size image

Microbial PUFA metabolites and adipose inflammatory response

We also examined the influence of HYA- or LA-feeding on host lipid metabolism over a period of 12 weeks. Interestingly, long-term HFD-feeding for 12 weeks compared with NC feeding resulted in elevated KetoB and HYB levels in the cecum relative to levels observed resulting from HFD-feeding for 2 weeks, whereas long-term HFD-feeding for 12 weeks, similar to HFD-feeding for 2 weeks, resulted in decreased HYA levels (Supplementary Fig. 3a). In addition, long-term HFD-feeding for 12 weeks, similar to HFD-feeding for 2 weeks, suppressed Cla-hy expression, whereas long-term HFD-feeding for 12 weeks increased Cla-dh and Cla-er expression as compared with HFD-feeding for 2 weeks (Supplementary Fig. 3b). Examination of the 16S gene-base cecal microbiome showed that long-term HFD-feeding, similar to LA-supplemented HFD feeding for 2 weeks, increased the abundance of the Lactobacillaceae family relative to that in controls (Supplementary Fig. 3c). Interestingly, we found that HYA supplementation increased Lactobacillaceae abundance to a greater extent than LA supplementation (Supplementary Fig. 3c), with hierarchical clustering of individual families confirming the effect of HFD, LA-supplemented HFD, and HYA-supplemented HFD feeding on the gut microbiome (Supplementary Fig. 3d). LCFAs, such as PUFAs, are mainly absorbed in the small intestine; therefore, we next examined the tissue-transferred FA profile in the ileum by lipid-metabolome analysis, finding that HYA supplementation increased KetoA levels in LA-derived gut microbial metabolites in the ileum (Fig. 4a) and significantly increased HYA levels in ileum and plasma (Fig. 4b). Subsequently, we found that LA-supplementation increased the abundance of FA metabolites related to the arachidonic acid (AA) cascade as compared with that observed in control mice, although levels of these FA metabolites in HYA-fed mice were similar to those in control mice or slightly decreased (Fig. 4a). Moreover, based on quantitative analysis in the ileum, AA and prostaglandin E2 (PGE2) levels in LA-fed mice were significantly increased as compared with those in control mice, whereas levels in HYA-fed mice were similar to those in control mice (Fig. 4c). Since prostaglandins and thromboxane via the AA cascade are considered to be lipid mediators of the inflammatory response25, we examined whether LA supplementation augmented the adipose inflammation response via the AA cascade. As anticipated, we found a significant increase in F4/80-positive macrophages in the WAT of LA-fed mice as compared with that of HYA-fed mice (Fig. 4d). In addition, adipose PGE2 levels in LA-fed mice were significantly higher than those in control mice, whereas comparable levels were observed between control and HYA-fed mice (Fig. 4e). Furthermore, we found that the expression of F4/80, the inflammatory marker tumor necrosis factor α (TNF-α), and monocyte chemoattractant protein-1 (MCP-1; also known as CCL2) was markedly increased in LA-fed mice as compared with control and HYA-fed mice (Fig. 4f). Therefore, in contrast to HYA supplementation, LA supplementation promoted the progression of adipose inflammation via the AA cascade.

Fig. 4 Gut microbial PUFA metabolites and adipose inflammatory response. a Heat map of FA profiles in the ileum (n = 4 per group). b HYA was detected in the ileum (left) and plasma (right) (n = 9 per group in the ileum; n = 9, 8, and 9 per group in the plasma). c Arachidonic acid (left) and PGE2 (right) were quantified in the ileum (n = 9 per group for arachidonic acid; n = 8, 9, and 9 per group for PGE2). d WAT sections were labeled by F4/80 (green), caveolin-1 (red), and DAPI (blue), and F4/80-positive cells were measured (n = 7 per group). Scale bar, 400 μm. e Levels of adipose PGE2 (n = 10 per group). f mRNA expression of F4/80, Tnfα, and Mcp1 in the WAT of HFD-induced obese mice (n = 10 per group). **P < 0.01; *P < 0.05 vs. control (Tukey–Kramer test). ##P < 0.01; #P < 0.05 vs. HYA (Tukey–Kramer test). Results are presented as means ± SE. Source data are provided as a Source Data file 4 Full size image

HYA directly regulates glucose homeostasis and GLP-1 release

We then investigated the effects of the gut microbial PUFA-metabolite HYA on GLP-1 secretion and glucose homeostasis. The incretin GLP-1, gut hormone that stimulates glucose-induced insulin secretion and inhibits food intake, is secreted from enteroendocrine L cells, which are primarily found in the ileum and colon26. Following oral administration of HYA (1 g/kg), HYA concentrations peaked 1 h after administration in plasma and the ileum, and 3 h after administration in the colon (Fig. 5a). In addition, HYA administration increased gut microbial HYA metabolites, such as HYC, KetoA, and KetoC, as well as HYA concentration in the ileum (Supplementary Fig. 4a). Similar to the peak time in the ileum, we found that plasma GLP-1 levels following both HYA and LA administration also peaked 1 h after administration, with the peak plasma GLP-1 levels following HYA administration higher than those in controls and LA-administered mice from 0.5 h to 3 h (Fig. 5b). Therefore, we performed oral GTT (OGTT) and intraperitoneal GTT (IPGTT) based on increases in GLP-1 levels following HYA administration at 1 h. Following oral administration of LA or HYA and after oral or intraperitoneal administration of glucose 2-h later, we found that HYA administration significantly suppressed increases in blood glucose as compared with that found in control and LA-administered mice (Fig. 5c and Supplementary Fig. 4b). Moreover, plasma insulin levels following glucose administration peaked at 30 min, and glucose-induced insulin secretion levels in HYA-administered mice were higher than those in control and LA-administered mice (Fig. 5d). Next, we examined whether HYA induces GLP-1 secretion using the mouse intestinal secretin tumor-cell line STC-1. We found that the LA-derived gut microbial PUFA metabolites KetoA, KetoC, and HYA, in particular, strongly induced GLP-1 secretion in a dose-dependent manner in STC-1 cells, whereas HYB and KetoB hardly induced these effects (Fig. 5e). In addition, LA-derived gut microbial PUFA metabolites including HYA, exhibited similar effects in GLUTag cells, an intestinal murine L-cell line (Supplementary Fig. 4c). Under physiological conditions, although HYA levels were the highest among HYA, HYC, KetoA, and KetoC levels in plasma, we found that the levels of all of them were markedly lower than those of LA in plasma (Fig. 5f). Our findings indicated that acute administration of HYA promoted GLP-1 secretion in the intestinal environment and improved glucose homeostasis.

Fig. 5 HYA directly regulates glucose homeostasis and GLP-1 release. Individual FAs (HYA and LA; 1 g/kg) were administered by gavage, followed by a HYA quantification in plasma (left), ileum (center), and colon (right) (n = 8 animals). b Time-course changes in plasma GLP-1 from the tail vein was measured after oral administration of FAs (n = 7 animals per group). Basal GLP-1 concentration at time 0 was set as 100%. c OGTT was analyzed 2 h after individual FA administration (HYA and LA; 1 g/kg) by gavage (n = 8 animals per group). d Individual FAs were administered, and 2 h later, time-course changes in plasma insulin from the tail vein were measured after oral administration of glucose (n = 8 animals per group). **P < 0.01; *P < 0.05 vs. control (Tukey–Kramer test). ##P < 0.01; #P < 0.05 vs. HYA (Tukey–Kramer test). Results are presented as means ± SE. e STC-1 cells were treated with LA-derived gut microbial metabolites [in a dose-dependent manner (20, 100, and 200 μM)], and GLP-1 concentration was measured in culture medium (n = 6 independent cultures from three biological replicates). **P < 0.01; *P < 0.05 vs. None (Tukey–Kramer test). f LA-derived gut microbial metabolites were detected in plasma of NC-fed mice (n = 8 animals). Results are presented as means ± SE. Source data are provided as a Source Data file 5 Full size image

HYA contributes to host metabolic condition via GPCRs

We further investigated HYA-mediated GLP-1 secretion signaling. A previous study reported that PUFAs promote GLP-1 secretion via GPR40 and GPR120 as FFARs27. Using a heterologous expression system, we found that HYA, KetoA, and KetoC rather than HYC, HYB, or KetoB strongly increased [Ca2+]i levels as compared to LA in both GPR40- and GPR120-overexpressing HEK293 cells, whereas these effects were not observed in doxycycline (−) control HEK293 cells (Fig. 6a, b). Treatment of STC-1 cells with small-interfering (si)RNAs for GPR40 and GPR120 (Supplementary Fig. 5) significantly inhibited HYA- or LA-induced GLP-1 secretion, whereas HYB exhibited no effects (Fig. 6c). We then examined the intracellular signaling mechanism whereby HYA mediates GLP-1 secretion through GPR40 and GPR120. Because GPR40 and GPR120 couple with Gq, these receptors elevate the level of [Ca2+]i and activate phospholipase C (PLC)28. We found that HYA- or LA-mediated GLP-1 secretion was effectively blocked by treatment with the mitogen-activated protein kinase (MAPK) kinase inhibitor U0126 (Fig. 6d) and the PLC inhibitor U73122 (Fig. 6e) but not with the CaM kinase II inhibitor KN-62 (Fig. 6f). Therefore, HYA-mediated GLP-1 secretion was mediated through a GPR40– and GPR120–PLC–MAPK cascade rather than via Ca signaling. Next, we generated GPR40- and GPR120-deficient mice using the CRISPR/Cas9 system in order to examine the effects of HYA via GPR40 and GPR120 in vivo (Supplementary Fig. 6a–c and Supplementary Fig. 7a–c). As expected, we found that the HYA-induced increase in GLP-1 secretion in wild-type mice was abolished in both GPR40- and GPR120-deficient mice (Fig. 6g). Moreover, suppression of the increase in blood glucose level following glucose administration by HYA pre-oral administration in wild-type mice was also abolished in both GPR40- and GPR120-deficient mice (Fig. 6h). Therefore, HYA administration promoted GLP-1 secretion and improved glucose homeostasis via activation of GPR40 and GPR120. In addition, we examined the mechanism of HYA-mediated suppression of lipid absorption in the gut. The PGE2 regulates intestinal peristalsis via the EP3 receptor29, and administration of an EP3 agonist promotes intestinal peristalsis (Supplementary Fig. 8a). Interestingly, we found that HYA was a low-affinity ligand for EP3, and that the observed effect of its administration was higher as compared with that observed with LA (Supplementary Fig. 8b). Moreover, we found that HYA promoted Gq-coupled EP3-mediated elevation in [Ca2+]i in EP3-overexpressing Chem-1 cells. Furthermore, oral administration of HYA and LA significantly promoted intestinal peristalsis as compared with that observed in controls, with the effect of HYA greater than that of LA (Supplementary Fig. 8c). In the presence of piroxicam, the addition of an EP3 agonist induced frequent contractions that mimicked spontaneous giant contractions (GCs), and we found that the frequency and amplitude of HYA-induced GC-like contractions increased in a dose-dependent manner, with these effects blocked by treatment with an EP3 antagonist (Supplementary Fig. 8d). Our findings indicated that HYA promoted intestinal peristalsis by acting as a low-affinity EP3 agonist to suppress lipid absorption in the gut.

Fig. 6 HYA contributes to host metabolic condition via GPR40 and GPR120. Mobilization of [Ca2+]i induced by LA-derived gut microbial metabolites was monitored in Flp in a hGPR40 or b hGPR120 T-REx HEK293 cells. Data are presented as Ca2+ intensity. Cells were cultured for 24 h and then treated with or without 10 μg/mL doxycycline (n = 8 independent cultures with doxycycline from three biological replicates; n = 6 independent cultures without doxycycline from two biological replicates). Closed symbols represent values from cells treated with doxycycline, and open symbols denote untreated groups. c–f The inhibitory effects of c Gpr40 and Gpr120 siRNA, d MEK inhibitor (U0126), e PLC inhibitor (U73122), and f CaMKII inhibitor (KN-62) on GLP-1 secretion following LA, HYA, or HYB treatment (n = 4 independent cultures from two biological replicates). **P < 0.01 vs. None (Tukey–Kramer test). ##P < 0.01; #P < 0.05 vs. LA (Tukey–Kramer test). $$P < 0.01 vs. HYA (Tukey–Kramer test). (−) represents untreated cells with siRNA or antagonist. Results are presented as means ± SE. g GLP-1 concentration and h OGTT in wild-type (left, n = 10 animals per group), Gpr40-deficient (middle, n = 8 animals per group), and Gpr120-deficient (right, n = 9, 10, and 9 animals per group) mice were analyzed 2 h after FA administration. **P < 0.01 vs. Control (Tukey–Kramer test). #P < 0.05 vs. LA (Tukey–Kramer test). (−) represents the mice without FA administrations. Results are presented as means ± SE. Source data are provided as a Source Data file 6 Full size image

HYA (+) Lactobacillus contributes to host metabolic benefits

We then performed microbial transplantation experiments using HYA-producing gut microbes in order to clarify whether HYA production by gut microbiota contributes to host metabolic improvement. Based on HYA yield (Supplementary Table 1), we selected L. salivarius (JCM1044, JCM1042, and JCM1231) as the HYA (+) strain and L. johnsonii (JCM1022 and JCM8791) and L. acidophilus (JCM1229) as HYA (−) strains and confirmed their intestinal-bacterial colonization (Supplementary Fig. 9a, b). Five weeks after colonization, we found that the fecal HYA levels in HYA (+)-colonized mice were significantly higher than that in GF mice, with no discernable change in this level observed between groups (Supplementary Fig. 9c). After feeding an HFD to Lactobacillus-colonized 4-week-old mice for 12 weeks, we found that body weight during growth and fat mass at 16 weeks of age in HYA (+)-colonized mice significantly decreased as compared with those in HYA (−)-colonized mice (Fig. 7a, b). Similar to our observations in HYA-supplemented HFD-fed mice, plasma glucose levels in HYA (+)-colonized mice were also significantly lower than those in HYA (−)-colonized mice (Fig. 7c), whereas total cholesterol and triglyceride levels were similar between these groups (Fig. 7d, e). Moreover, GLP-1 levels were significantly higher in HYA (+)-colonized mice relative to those in HYA (−)-colonized mice (Fig. 7f), whereas plasma insulin levels in HYA ( + )-colonized mice were significantly lower than those in HYA (−)-colonized mice (Fig. 7g). In addition, we found significantly decreased adipose Tnfα expression and increased fecal triglycerides in HYA (+)-colonized mice relative to these levels in HYA (−)-colonized mice (Fig. 7h, i), and that glucose clearance as assessed by OGTT was improved in HYA (+)-colonized mice as compared with HYA (−)-colonized mice (Fig. 7j). Furthermore, cecal HYA levels in HYA (+)-colonized mice were significantly higher than those in GF and HYA (−)-colonized mice and similar to those in conventionally raised mice (Fig. 7k). Therefore, we found that HYA-producing gut microbial Lactobacilli improved metabolic conditions. In addition, although in fecal microbiota-transplantation (FMT) experiments, OGTT was significantly improved in FMT mice from long-term HYA-supplemented HFD-fed mice as compared with FMT mice from control HFD-fed mice (Supplementary Fig. 9d), HYA-supplemented HFD feeding also significantly suppressed weight gain as compared with control HFD feeding during growth under metabolic cage bleeding in order to exclude the influence of coprophagy (Supplementary Fig. 9e). These results indicated that in addition to HYA direct effects, HYA-mediated changes in gut microbial composition also influenced to the host metabolic condition. Collectively, our results indicated that gut microbiota conferred host resistance to obesity by metabolizing dietary PUFAs into HYA, which functioned to improve host metabolic homeostasis.