Gut microbiota-derived tryptamine and phenethylamine impair insulin sensitivity in metabolic syndrome and irritable bowel syndrome

Irritable bowel syndrome (IBS) is a common functional bowel disorder characterized by bowel habit changes and recurrent abdominal pain. Recent studies reveal a link between IBS and a higher prevalence of metabolic syndrome and type 2 diabetes (T2D), suggesting IBS as a risk factor for metabolic disorders. However, the underlying mechanisms remain unclear. The gut microbiome is causally linked to both metabolic dysfunctions and gastrointestinal disorders, suggesting that gut dysbiosis in IBS could contribute to metabolic syndrome development. Changes in gut microbiota composition in metabolic disorders lead to altered levels of gut microbial products like lipopolysaccharides, short-chain fatty acids, bile acids, and others. These changes impact glucose tolerance. Dietary amino acids, including aromatic and branched-chain amino acids, are catabolized by the gut microbiota into metabolites that can affect metabolic health. Previous research identified *Ruminococcus gnavus*, an anaerobic bacterium associated with IBS, as a producer of tryptamine and phenethylamine from dietary tryptophan and phenylalanine. *R. gnavus* has also been linked to features of metabolic syndrome, but its causal role and underlying mechanisms remain unknown. This study aimed to investigate the mechanistic contribution of gut microbe-derived tryptamine and phenethylamine to the development of insulin resistance in metabolic syndrome and IBS.

The existing literature extensively documents the correlation and causality between the gut microbiome and metabolic disorders, including metabolic syndrome, insulin resistance, and diabetes, as well as gastrointestinal disorders such as IBS. Studies have shown that alterations in gut microbiota composition can significantly influence the levels of various microbial metabolites, which in turn affect glucose tolerance and metabolic health. The role of dietary amino acid metabolism by gut microbiota and the subsequent impact of the resulting metabolites on host metabolism have also been investigated. Previous research has highlighted the production of tryptamine and phenethylamine by *R. gnavus*, linking it to diarrheal symptoms in IBS patients. However, a comprehensive understanding of the causal relationship between *R. gnavus*, its metabolites, and the development of insulin resistance, particularly in the context of IBS and its co-occurrence with metabolic syndrome, was lacking before this study. This research builds upon existing knowledge by investigating the precise mechanisms through which *R. gnavus*-derived tryptamine and phenethylamine contribute to the pathogenesis of insulin resistance.

This study employed a multi-faceted approach involving human subjects, monkey models, and in vitro experiments. **Monkey Study:** Two crab-eating macaque studies were conducted. The first metabolomics study (Yunnan Yinmore Biotechnology company ethical approval No. YMB1704) involved categorizing age-matched monkeys into normal, pre-diabetes, and diabetes groups based on fasting blood glucose (FBG) and HbA1c levels. Serum and fecal samples were collected for analysis. The second study (Huazhen Biosciences company ethical approval No. HZ2021047) assessed the impact of tryptamine treatment on biochemical parameters, including oral glucose tolerance test (OGTT) and serum insulin levels. **Mouse Study:** All mouse studies were approved by the Hong Kong Baptist University's Committee on the Use of Human & Animal Subjects in Teaching & Research, following ARRIVE guidelines. Male C57BL/6J mice were used. Antibiotics-treated mice were generated using antibiotic cocktails. Germ-free mice were utilized for monoassociation studies. *Taar1* knockout mice were used to investigate the role of TAAR1 in the observed effects. **Human Study:** Three human cohort studies were conducted, each with ethical approvals and informed consent obtained from participants. The first study compared healthy controls and IBS patients, the second compared healthy controls and T2D subjects, and the third (GUT2D study, ChiCTR-TRC-14004959) involved a dietary fiber intervention in T2D patients. **Cell Study:** 3T3-L1 adipocytes were used for in vitro studies of insulin signaling and glucose uptake. The effects of tryptamine, phenethylamine, and related compounds on insulin signaling were assessed. ERK inhibitors and TAAR1 antagonists were employed to investigate the mechanisms. **Bacterial Strain Culture:** A tryptamine-producing *Lactobacillus casei* strain was engineered by incorporating the tryptophan decarboxylase (TDC) gene from *Ruminococcus gnavus*. This strain and a vector-only *L. casei* were used to investigate the impact of TDC-produced tryptamine in vivo. **Analytical Techniques:** LC-MS was used for targeted metabolomics, measuring tryptamine, phenethylamine, and related metabolites in various samples (serum, feces, culture medium, and tissues). Western blotting was used to assess protein phosphorylation levels. Phosphoproteomics analysis was conducted to identify proteins differentially expressed in response to tryptamine treatment. **Statistical Analysis:** Appropriate statistical tests (t-tests, ANOVA, correlation analysis) were applied depending on the nature of the data.

This study revealed several key findings: 1. **Association between *R. gnavus*, tryptamine/phenethylamine, and insulin resistance:** In both IBS patients and healthy controls, levels of *R. gnavus*, tryptamine, and phenethylamine were positively correlated with the TyG index, a marker of insulin resistance. IBS patients exhibited significantly higher levels of FBG, TG, and TyG compared to healthy controls. 2. **Causal role of *R. gnavus* in insulin resistance:** Monoassociation of *R. gnavus* in germ-free mice resulted in impaired glucose tolerance and reduced insulin sensitivity, accompanied by increased fecal tryptamine and phenethylamine. Colonization with a *L. casei* strain ectopically expressing the *R. gnavus* TDC gene produced similar results, confirming the causal role of tryptamine/phenethylamine. 3. **Tryptamine/phenethylamine and glucose intolerance in T2D:** Fecal tryptamine and phenethylamine levels were significantly higher in T2D patients compared to healthy controls. These metabolites were positively correlated with fasting blood glucose. Similar results were observed in monkeys with spontaneous metabolic syndrome. 4. **Dietary fiber intervention reduces tryptamine/phenethylamine:** In a dietary fiber intervention study in T2D patients, the reduction of *R. gnavus* was associated with decreased fecal tryptamine and phenethylamine. This reduction correlated positively with improvements in HbA1c and HOMA-IR indexes. 5. **Direct effects of tryptamine/phenethylamine on insulin signaling:** In vivo administration of tryptamine and phenethylamine to healthy mice and monkeys impaired glucose tolerance and insulin sensitivity, suppressing insulin-induced Akt phosphorylation in metabolic tissues. In vitro studies using 3T3-L1 adipocytes confirmed the direct inhibitory effects of tryptamine and phenethylamine on insulin signaling. 6. **TAAR1-ERK signaling axis:** Phosphoproteomics analysis revealed ERK activation in insulin-sensitive tissues after tryptamine treatment. ERK inhibitors significantly improved glucose intolerance and insulin resistance in tryptamine- and phenethylamine-treated mice, reversing the inhibitory effects on Akt phosphorylation. TAAR1 antagonism (using EPPTB) also improved glucose tolerance and insulin sensitivity, and downregulated ERK phosphorylation. Genetic ablation of *Taar1* provided further support for the role of the TAAR1-ERK axis. 7. **TAAR1 inhibition alleviates insulin resistance induced by gut dysbiosis:** Pharmacological TAAR1 antagonism using EPPTB partially abrogated *R. gnavus*-induced insulin resistance in antibiotic-treated mice. Fecal microbiota transplantation from IBS patients with high tryptamine/phenethylamine levels induced glucose intolerance in antibiotic-treated mice, which was also significantly improved by *Taar1* genetic ablation.

This study provides strong evidence for a causal link between *R. gnavus*-derived tryptamine and phenethylamine and insulin resistance in both IBS and metabolic syndrome. The use of both human and monkey models, the latter minimizing confounding variables often present in human studies, strengthens the results. The findings demonstrate that tryptamine and phenethylamine impair insulin signaling through the TAAR1-ERK pathway. The success in partially reversing insulin resistance with TAAR1 antagonism suggests this pathway as a potential therapeutic target for managing metabolic syndrome related to gut dysbiosis. Future research should focus on developing TAAR1 modulators as potential therapeutic agents. The study also highlights the complex interplay between gut microbiota, their metabolites, and host metabolic pathways. Further research should investigate the long-term effects of TAAR1 modulation and the potential for personalized interventions based on gut microbiome profiles.

This research demonstrates a novel pathogenic mechanism underlying the increased prevalence of metabolic syndrome in IBS patients. *Ruminococcus gnavus*-derived tryptamine and phenethylamine impair insulin sensitivity by activating the TAAR1-ERK signaling pathway. These findings highlight the TAAR1 signaling axis as a potential therapeutic target for managing gut dysbiosis-induced metabolic syndrome. Further studies should investigate the development of TAAR1 modulators and the potential for personalized interventions based on individual gut microbiome profiles.

While this study provides compelling evidence for the role of tryptamine and phenethylamine in insulin resistance, some limitations exist. The study focused primarily on *R. gnavus*; other bacteria may also contribute to insulin resistance through similar or different mechanisms. The dietary fiber intervention study had a relatively short duration (84 days). Longer-term studies are needed to evaluate the sustained effects of interventions targeting the gut microbiome and its metabolites. Further research is required to determine the optimal dosage and long-term safety of TAAR1 modulators.