Microbiome | Wesfarmers reveals the mechanism by which high-fat diet disrupts peripheral tryptophan-kynurenine metabolic homeostasis

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Clumsy is better than cleverness, simplicity is better than gorgeousness. When I entered the graduate school five years ago, I started the research on the relationship between the microbiome and the health of the host. In a trance, until now, along the way, although the road is also bumpy, fortunately, there are always good teachers and helpful friends to help. Professor Zheng Wei from the School of Resources and Environment, Northwest A&F University, is a teacher and even a brother. He led me to the beginning and led me to carry out data analysis work. Professor Liu Yongxin from the Food Center of Shenzhen Institute of Genomics, Chinese Academy of Agricultural Sciences is a scholar who is knowledgeable and willing to share. Although we met by chance, he gave me the most selfless help. The development of all the work is inseparable from the help and support of my teacher's wife, Professor Chai Xuejun. My teacher's wife's love and pursuit of scientific career have deeply affected me. My supervisor, Professor Zhao Shanting, gave me the most solid understanding and support during my scientific research process. Teacher Zhao's tolerance and help to me are the driving force for me to come to the present. Thanks to Professor Zhu Xiaoyan for his help and guidance during my study and work. Teacher Zhu's tireless scientific research attitude is a role model for me to learn from. I also sincerely thank the knowledge sharers for their contributions (thanks to Listenlii, Little White Fish's Biological Notes, Red Queen Academic, Weishengxin Biology, VisualHub and other official account bloggers for their sharing and help). Although there are thousands of words, it is difficult to express one ten-thousandth of the gratitude in my heart.

Gut microbiota-colonocyte interactions perturbed by a high-fat diet lead to dysregulation of peripheral tryptophan-kynurenine metabolism

High-fat diet-disturbed gut microbiota-colonocyte interactions contribute to dysregulating peripheral tryptophan-kynurenine metabolism

Article，2023-07-19

Microbiome, [IF 15.5]

DOI：https://doi.org/10.1186/s40168-023-01606-x

Original link : https://microbiomejournal.biomedcentral.com/articles/10.1186/s40168-023-01570-6

First author : Penghao Sun (孙梁赵); Mengli Wang (王梦丽)

Corresponding authors : Xuejun Chai (柴学军); Xiaoyan Zhu (Zhu Xiaoyan); Shanting Zhao (赵善庭)

Co-authors : Yong-Xin Liu (刘永新); Luqi Li (李琳琪); Wei Zheng (郑伟); Shulin Chen (陈林林)

Main unit :

Veterinary Medicine (College of Veterinary Medicine, Northwest A&F University, Yangling, 712100, Shaanxi, China; Life Science Research Core Services, Northwest A&F University, Yangling, 712100, Shaanxi, China; , Yangling, 712100, Shaanxi, China)

Shenzhen Branch, Guangdong Laboratory of Lingnan Modern Agriculture, Genome Analysis Laboratory of the Ministry of Agriculture and Rural Affairs, Agricultural Genomics Institute at Shenzhen, Chinese Academy of Agricultural Sciences, Shenzhen, Guangd ong 518120, China)

Xi'an Medical College (College of Basic Medicine, Xi'an Medical University, Xi'an, 710000, Shaanxi, China)

- Summary-

Abnormalities in tryptophan (Trp)-kynurenine (Kyn) metabolism have been implicated in the pathogenesis of human diseases. In particular, Kyn was found to be excessive in plasma in people on a long-term Western-style diet. Host-gut microbiota interactions are governed by diet and are critical for maintaining host metabolic homeostasis. However, the role of gut microbiota-colonocyte interactions perturbed by a Western diet in Trp metabolism remains to be elucidated. In this paper, 4-week-old mice were fed a high-fat diet (HFD) representative of a typical Western diet for 4 weeks, and a multi-omics approach was used to determine that HFD interferes with gut microbiota-colonocyte interactions leading to serum Trp-Kyn metabolism. The mechanism of the obstacle. Our results suggest that colonocyte-microbiota interactions dominate peripheral Kyn homeostasis in HFD mice. Mechanistically, sustained HFD disrupts mitochondrial energy homeostasis in colonocytes, which increases colonic epithelial oxygenation and induces metabolic reprogramming within the colon, ultimately leading to the expansion of the Proteobacteria phylum in the lumen of the colon. Lipopolysaccharide (LPS) derived from Proteobacteria stimulates colonic immune responses and upregulates the indoleamine-2,3-dioxygenase 1 (IDO1)-mediated Kyn pathway, leading to Trp depletion and Kyn accumulation in the peripheral circulation , which was further confirmed by transplantation of Escherichia coli (E.coli) indicator strains and inhibition of colonic IDO1 activity. Supplementation with butyrate promoted the function of mitochondria in colonocytes, thereby reshaping the gut microbiota in HFD mice, thereby improving Kyn accumulation in serum. Our results suggest that HFD disrupts the peripheral Kyn pathway in a gut microbiota-dependent manner and that the ongoing balance of gut bacteria-colonocyte interactions plays a central role in regulating host peripheral Trp metabolism. At the same time, this study provides new ideas for the treatment of metabolic disorders associated with the Western diet.

- Introduction -

Tryptophan (Trp) is an essential amino acid obtained entirely from the diet. Tryptophan and its metabolites play key roles in various physiological activities ranging from cell proliferation to coordinating the body's physiological balance. The concentration of free Trp in organisms is affected by the activity of several Trp metabolic pathways. About more than 95% of free Trp is metabolized through the kynurenine (Kyn) pathway, and its metabolites are involved in inflammation, immune response, and excitatory neurotransmission. Aberrant activation of peripheral Kyn pathways is thought to be associated with the onset and development of a variety of psychiatric and psychological disorders, such as depression and schizophrenia. In addition, due to the intricate relationship between Kyn metabolites and immune responses, Kyn has gradually been recognized as a mediator of various diseases such as inflammatory bowel disease, obesity, and cancer.

The gut commensal microbiota is a key regulator of physiological homeostasis in humans. Among a range of regulatory processes, many are mediated by microbe-derived metabolites or by environmental and host molecules transformed by microbes. More and more evidence shows that Trp plays a pivotal and unique role in a series of metabolites that constitute the two-way communication between gut microbes and hosts. At the same time, a large body of evidence suggests that the increased concentration of Kyn in the blood circulation may be due to the upregulation of indoleamine 2,3-dioxygenase 1 (IDO1), a rate-limiting enzyme in the Kyn pathway, mainly in the immune system. Expressed in systemic and intestinal mucosal tissues. The role of gut bacteria in controlling intestinal IDO1 activity has been demonstrated in germ-free mice. In addition, some gut bacteria encode enzymes that are homologous to the eukaryotic Kyn pathway and are thus able to produce Kyn and downstream metabolites such as 3-hydroxykynurenine (3-HK), which readily cross the blood-brain barrier and exhibited neurotoxic effects. However, the effect of compositional changes in the gut microbiota on Trp metabolism has not been fully elucidated.

Diet is one of the core factors affecting human health and the cause of many non-communicable chronic diseases. Over the past few decades, a high-fat, high-sucrose, low-fiber dietary pattern (also known as a Western diet) has become increasingly common throughout the world. A solid evidence of the impact of changing dietary patterns on human health is the rising incidence of metabolic diseases during the transition from traditionally non-industrial regions to Western societies. Cross-regional studies of the human microbiota have shown that dietary habits have a significant impact on the composition and richness of the gut microbiota, and that different dietary components shape the gut microbiome in a time-dependent manner. More and more studies have shown that almost all diet-related chronic diseases are related to the microbiome, which proves that the microbiome is a carrier and risk factor that mediates the occurrence and development of diet-related diseases. For example, human studies and animal models have shown that a high-fat diet (HFD) can affect the pathogenesis of gut microbes, thereby exacerbating chronic inflammation and the severity of inflammatory diseases. However, the role of gut bacteria in diet-induced systemic metabolic disturbances, especially amino acid metabolism, is becoming clearer but remains poorly understood.

This study aimed to investigate the mechanistic link between gut microbiota dysbiosis and Trp-Kyn metabolic disturbance using an animal model of HFD. Consistent with human epidemiological findings, this study found that long-term high-density lipoprotein cholesterol (HFD) disturbs the metabolism of Trp in serum, manifested by Trp depletion and upregulation of the Kyn pathway, which is consistent with the expansion of the proteobacteria in the colon closely related. Antibiotic treatment abolishes dysregulated Kyn metabolism in mice fed a high-fiber diet, but transfers it to mice fed a standard diet by fecal microbiota transplantation (FMT), suggesting that the gut microbiota plays an important role in high-fiber diet-fed mice Dysregulated Trp metabolism in vivo plays a causal role. Given the important role of IDO1 in the Trp-Kyn pathway, we hypothesized that HFD-altered interactions between the gut microbiota and colonocytes would contribute to the HFD-induced disturbance of Trp metabolism. Colonic RNA-sequencing analysis revealed that lipopolysaccharide (LPS) derived from Proteobacteria elicited a marked colonic inflammatory response, upregulated the expression of IDO1 in colonic tissue, and thus increased serum Kyn concentration, which was obtained by transplanting an indicator strain of Escherichia coli and depletion of colonic IDO1 was further confirmed. Having identified the central role of gut bacteria in regulating systemic Trp-Kyn metabolism, we sought to determine the underlying mechanisms of HFD-induced Proteobacteria overgrowth, a hallmark of gut dysbiosis. Our results showed that HFD decreased the concentration of bacteria-derived butyrate in the colon, which is the main energy source of colonocytes, and simultaneously upregulated long-chain and very long-chain fatty acid β-oxidation in colonocytes, triggering Oxidative stress in colon tissue, which impairs mitochondrial function. Disrupted mitochondrial bioenergetics destabilizes colonic epithelial hypoxia, increases luminal oxygen availability, and causes colonocyte metabolism to be redirected towards glycolysis, leading to increased lactate release and nitrate synthesis. These all provide additional respiratory electron donors or acceptors for the reproduction of Proteobacteria. At the same time, we also noticed that dietary butyrate supplementation reversed HFD-induced mitochondrial bioenergetics impairment and subsequent Trp-Kyn metabolic dysregulation caused by gut microbiota dysbiosis. Taken together, our findings highlight the causal role of the gut microbiota in diet-induced Trp metabolic disturbances and reveal the contribution of gut microbiota-colonocyte interactions in systemic metabolic homeostasis.

- result -

① HFD interferes with serum metabolic profile

HFD disturbed the serum metabolic profile

To reveal the effect of a Western-style diet on whole-body metabolic homeostasis, we fed 4-week-old mice with a high-fat diet (HFD) for 4 weeks to simulate continuous Western-style diet. We performed an untargeted metabolomics study on the serum of 8-week-old C57BL/6 mice fed standard chow (Chow) and HFD (Fig. 1a). Using UHPLC-HESI-HRMS-based untargeted metabolomics and Tidymass-based comprehensive computational framework, we detected 3135 ion features. We performed a global examination of metabolites using principal component analysis (PCA) and found that dietary patterns resulted in distinct serum metabolic profiles (P = 0.003) (Fig. 1b). We then performed a statistical evaluation of the ion feature matrix and screened out 614 significant features (P < 0.05) after removing redundant annotated metabolites. Chemical similarity enrichment analysis (ChemRICH) was performed to identify metabolite classes altered by HFD (Fig. 1c). In the chromatograms of the nodes, purple indicates that among the metabolites significantly changed between the Chow group and the HFD group, the metabolites enriched in the HFD group over the Chow group and/or reflect a more significant fold change. Forty-one chemicals were clustered, and metabolites altered by HFD mainly involved lipids and amino acids (Fig. 1c). To describe metabolites altered by HFD from the perspective of biological pathways, we performed quantitative metabolite set enrichment analysis (qMSEA) and found that HFD significantly affected whole-body Trp metabolism (Fig. 1d). Notably, HFD significantly upregulated the Kyn pathway in serum, as evidenced by elevated serum 3-hydroxykynurenine (3-HK) and Kyn concentrations and the Kyn/Trp ratio (Fig. 1e). These results suggest that sustained HFD perturbs Trp metabolism in serum, characterized by upregulation of the Kyn pathway.

Figure 1 HFD disrupts tryptophan metabolism. a Experimental workflow begins with animal dietary intervention and sample collection (n = 12 per group). b Principal component analysis (PCA) evaluating serum metabolome data comparing high-fat diet mice with standard diet mice (n = 6 per group). c Chemical similarity enrichment analysis (ChemRICH) clustered 614 HFD-altered serum metabolites by chemical similarity. d Quantitative metabolite set enrichment analysis (qMSEA) based on 99 metabolite sets associated with human metabolic pathways identified the top 25 serum metabolic pathways significantly disturbed by HFD (P < 0.05). e HFD significantly elevates peripheral Kyn metabolic pathways.

② Intestinal bacteria are related to serum Kyn concentration

Gut bacteria linked to serum Kyn concentration

The intricate relationship between gut microbiota and host metabolic balance prompted us to investigate whether observed changes in serum Trp metabolism were related to gut bacterial responses to dietary patterns. Consistent with previous reports, 16S rRNA sequencing of colonic contents revealed that continuous high-fat diet significantly reduced gut bacterial richness (Fig. 2a) and formed a distinct bacterial population relative to Zhou mice (PERMANOVA by Adonis, P = 0.001) (Fig. 2b). Furthermore, we noted that HFD mice exhibited an expansion of the Proteobacteria phylum (Fig. 2c), a hallmark of dysbiosis in the gut microbiota. To identify bacterial taxa altered by HFD, a linear discriminant analysis (LDA) effect size (LEfSe) approach was employed (Fig. 2e,f). The results showed that butyrate-producing bacteria, including Roseburia, Eubacterium_g8, Eubacterium_g23, and Eubacterium_g17, were reduced in HFD-fed mice, while bacteria associated with intestinal inflammation, such as Bilophila, Desulfovibrio, and Enterobacteriaceae, were abundant under sustained HFD Breeding (Fig. 2e,f). After correlating HFD-altered bacterial taxa with the disturbed serum metabolite matrix of HFD mice, we found that the abundance of HFD-enriched bacterial taxa correlated strongly with changes in serum metabolic profiles (Fig. 2g).

To further identify key bacterial taxa associated with the Kyn pathway, we regressed the abundance of LEfSe-discriminated bacterial taxa on serum Kyn concentrations using a random forest machine learning algorithm. Many studies have highlighted the link between Proteobacteria and metabolic diseases. One potential mechanism is that expansion of the Proteobacteria phylum activates intestinal mucosal immunity, leading to local and systemic inflammation and metabolic dysfunction. Likewise, we noted that HFD-enriched lipopolysaccharide (LPS) producers (mainly belonging to the Proteobacteria phylum) were significantly positively correlated with serum Kyn concentrations (Fig. 2h and S2a). These results suggest that HFD-induced gut microbiota dysbiosis, characterized by expansion of the Proteobacteria phylum, is closely associated with upregulation of the Kyn pathway in serum.

Figure 2 High-fat diet-induced intestinal flora dysbiosis is highly correlated with serum metabolic characteristics. a Gut bacterial α-diversity estimated by Shannon index (n = 8 per group). b Principal coordinates analysis (PCoA) plot showing differences in microbial composition. c Phylum level species composition. d Ratio of Firmicutes to Bacteroidetes. e Species cladograms generated from linear discriminant analysis effect sizes (LEfSe) showing the most divergent bacterial taxa in the colonic contents of Chow (green) or HFD (blue) mice (LDA value = 2.0; P < 0.05). g Shows pairwise comparisons of HFD-altered bacterial taxa, color gradients indicate Pearson correlation coefficients. HFD-altered serum metabolites (blue: HFD-enriched serum metabolites; green: down-regulated serum metabolites in HFD-fed mice) were associated with each bacterial taxa by Mantel test. h The top 10 bacterial biomarkers were identified by random forest regression of the relative abundance of LEfSe-determined bacterial taxa against serum Kyn concentrations. Statistical significance of selected bacterial biomarkers was assessed by permutation test (999 times).

③ HFD in a gut microbiota-dependent manner

Disruption of peripheral Kyn pathway

HFD disrupted the peripheral Kyn pathway in a gut microbiota-dependent manner

Given the link between gut bacteria and serum Kyn concentrations, we further explored the causal relationship between HFD-induced gut microbiota dysbiosis and peripheral Trp-Kyn pathway dysfunction. We transplanted the fecal microbiota of HFD-fed mice into standard diet-fed mice (C-FMT) and subsequently detected Kyn pathway metabolites in the sera of C-FMT mice (Fig. 3a). The efficiency of microbial colonization is closely related to the availability of niches in the environment. Therefore, to increase the efficiency of fecal microbiota transplantation (FMT), mice were treated with a cocktail of antibiotics (Abx) for 3 days prior to FMT to eliminate gut commensal flora. Our results showed that more than 80% of the native gut microbiota was cleared after 3 days of oral administration of Abx. After 4 weeks of continuous daily FMT administration, the microbial composition of C-FMT mice was more similar to that of HFD mice than that of Chow mice (Fig. 3b,c). At the phylum level, C-FMT mice had increased abundance of the Proteobacteria phylum compared with Chow mice (Fig. 3d,e). Meanwhile, the abundance patterns of major bacterial taxa identified by microbial metabolite association studies (Fig. 2g–h and S2) in C-FMT mice were consistent with those in HFD mice (Fig. 3f). These results demonstrate that the gut microbiota of standard diet-fed mice receiving FMT was significantly remodeled to more closely match the bacterial composition of HFD mice.

Next, we examined changes in serum metabolism of C-FMT mice by untargeted metabolomics and found that FMT donated by HFD mice significantly altered the metabolic profile of standard diet-fed mice, making it comparable to that of HFD mice. The spectra are closer (Fig. 3g). We further examined the metabolism of Trp-Kyn in serum and found depletion of Trp and increased 3-HK concentration and Kyn/Trp ratio in C-FMT mice (Fig. 3h). Based on the above results, we confirmed that the dysregulation of peripheral Trp-Kyn metabolism in HFD mice was at least partially attributable to diet-altered gut microbiota. Given the close relationship between dietary composition and individual metabolic profiles, we sought to determine whether HFD could guide systemic Trp-Kyn metabolism independently of the gut microbiota. To further confirm the role of gut microbiota in HFD-induced dysregulation of peripheral Trp-Kyn metabolism, we generated pseudogerm-free mice by injecting HFD-fed mice (H-Abx) with a combination of antibiotics (Fig. 3a). As shown in Figure 3i, the activity of the Kyn pathway was decreased in the serum of H-Abx mice compared with HFD mice, suggesting that the gut microbiota played a decisive role in mediating HFD-induced Trp-Kyn metabolic dysregulation. Taken together, these results highlight that HFD disrupts peripheral Trp-Kyn metabolism in a gut microbiota-dependent manner.

Figure 3 Causal role of gut microbiota in mediating HFD-induced dysregulation of peripheral tryptophan-kynurenine metabolism. a Fecal pellets were collected from 8-week-old HFD mice and then used for fecal microbiota transplantation ( FMT). Mice fed a standard diet were treated with antibiotics for 3 days and then underwent fecal microbiota transplantation (C-FMT) for 4 weeks. b, c PCoA showing the similarity of the microbiota composition of mice after FMT to that of donors (n = 8 per group). The taxonomic profile of gut bacteria in ef C-FMT mice was more similar to that of HFD mice. g PCA analysis was performed to evaluate the serum metabolic profile of standard diet-fed mice after receiving FMT from HFD mouse donations (n = 6 per group). FMT donated by hHFD mice enhanced the peripheral Trp-Kyn pathway in standard diet-fed mice. i Elimination of gut bacteria with antibiotics abolished HFD-induced dysregulation of peripheral Trp-Kyn metabolism.

④ Inhibition of IDO1 in colonocytes

May attenuate serum Kyn accumulation induced by intestinal dysbiosis

Inhibition of IDO1 in colonocytes attenuated gut dysbiosis-induced serum Kyn accumulation

Given the central role of IDO1 in regulating the Trp-Kyn pathway, we hypothesized that HFD-induced gut microbiota dysbiosis might overactivate IDO1 in the colon, thereby increasing serum Kyn concentrations. To investigate the effect of HFD on the interaction between the gut microbiota and colonocytes, we compared the gene expression profiles of the colon tissues of the Chow group and the HFD group using RNA-sequencing technology. There was a significant difference in the transcriptome of the colon tissue between the Chow group and the HFD group (P = 0.016) (Fig. S4a). Among 17,381 gene transcripts, we found 401 (|log2FC| > 1, P < 0.05) to be significantly associated with high-fat diet (Fig. 4a). Genome enrichment analysis (GSEA) showed that the "tryptophan metabolism" pathway was significantly upregulated in the colon tissue of HFD mice compared with Chow mice (P = 0.02), which was characterized by high expression of the IDO1 gene (Fig. 4b) . To further confirm the expression of IDO1 in colon tissue, we performed immunofluorescence staining and found that the expression of IDO1 was significantly increased in the colon of HFD mice (P < 0.001) (Fig. 4c). These results suggest that sustained HFD upregulates IDO1 in colonocytes. Given the stimulation of IDO1 by pro-inflammatory cytokines, we next investigated the immune response in the colon. Consistent with the results of Proteobacteria expansion in the colonic microbiota (Fig. 2c,e), the bacterial LPS-mediated inflammatory response was significantly activated in colonic tissue (P = 0.04) (Fig. 4d), and the concentration of LPS in colonic content increase also verified this (Fig. 4e).

To test the pathogenic role of Proteobacteria expansion in colonic IDO1 upregulation and subsequent serum Trp-Kyn metabolic disturbance, we isolated E. coli indicator strains from feces of HFD mice and enriched them in vitro (> 1 × 108 CFU/ml) in order to transplant the bacteria into standard diet-fed mice (Fig. 4f). Oral administration of E. coli for 3 days successfully colonized the colon lumen of mice on a standard diet (CE.coli). E. coli expansion significantly increased the concentration of LPS in the intestinal lumen (P < 0.001) (Fig. 4g) and the expression of IOD1 in the colon tissue (P < 0.001) (Fig. 4c, h), while the Kyn pathway was also upregulated in the serum ( Figure 4i). These results underscore the priming role of Proteobacteria expansion in HFD-induced Trp-Kyn metabolic disturbances. We further administered palmatine, an irreversible IDO1 inhibitor with very low oral bioavailability, to mice fed a standard diet to investigate the dominance of colonic IDO1 on peripheral Trp-Kyn metabolism (C-Pal). Some studies have shown that palmatine has an inhibitory effect on Gram-negative bacteria such as E. coli. Therefore, palmatine should be discontinued when mice received the indicator strain of E. coli isolated from hyperlipidemic mice (Fig. 4f). Similar to CE.coli mice, the late effect of palmatine did not affect E. coli colonization in the colon of C-Pal mice (Fig. S5a). Meanwhile, our results showed that oral administration of palmatine significantly inhibited colonic IDO1 in C-Pal mice (P < 0.001) (Fig. 4c,h) and inhibited the serum Kyn pathway (Fig. The concentration of LPS in mice was higher than that in Chow mice (Fig. 4g). Based on the above results, we concluded that the overgrowth of proteobacteria triggers the upregulation of IDO1 in the colon and plays a decisive role in the dysregulation of peripheral Trp-Kyn metabolism induced by HFD.

Fig. 4 Hyperactivation of indoleamine-2,3-dioxygenase 1 in colonocytes mediates dysregulation of peripheral Trp-Kyn metabolism caused by gut microbiota dysbiosis. a Volcano plot showing the HFD-altered transcriptome in colonocytes (n = 3 per group). b Genome Enrichment Analysis (GSEA) showing the enrichment of the Trp metabolic genome (left) and the heatmap of the genes involved (right) (n = 3 per group). d Enriched genome (left) and heatmap (right) of genes involved in lipopolysaccharide (LPS)-induced inflammation (3 per group). f Escherichia coli (E.coli) indicator strains were isolated from feces of HFD mice and enriched by bacterial transplantation in vitro to confirm the role of proteobacteria expansion in HFD-induced Kyn pathway dysregulation (CE.coli) causal effect. To determine the central role of colonic IDO1 in E. coli-mediated upregulation of the Kyn pathway, mice were administered palmatine to deplete colonic IDO1 before receiving E. coli (C-Pal). h IDO1 activity in the colon (9 sections from 3 mice). i E. coli transplantation upregulated the serum Kyn pathway, which was reversed by pretreatment with palmatine (6 sections per group).

⑤ HFD damages the mitochondrial function of colon cells

Increased accessibility of Proteobacteria to host-derived respiratory substrates

HFD-disrupted mitochondria in colonocytes promoting the accessibility of host-derived respiratory substrates to Proteobacteria

Colonocyte metabolism is critical for shaping the colonic microbiota. One such mechanism is that the host restricts the supply of oxygen and nitrate to the lumen of the colon, which favors the growth of obligate anaerobes specialized in fermentation. In turn, butyrate from obligate anaerobes activates peroxisome proliferator-activated receptor-γ (PPAR-γ), which promotes mitochondrial β-oxidation of short-chain fatty acids (SCFAs) in colonocytes to produce adenosine triphosphate ( ATP) to maintain energy metabolism. Given the significant changes in the transcriptional profile of colonic tissues (Fig. 4a and S4a), we speculated that HFD-induced colonocyte dysfunction might be one of the reasons for the overgrowth of Proteobacteria. Consistent with the reduction in the number of butyrate-producing bacteria (Fig. 2e,f), sustained HFD significantly reduced colonic butyrate concentrations (P < 0.01) (Fig. 5a), and butyrate provided more than 70% of the colonic epithelial cells. energy of. At the same time, we noticed increased long-chain and very long-chain fatty acid metabolic processes in colonocytes of mice fed a high-fat diet (Fig. S6a–b). Previous studies have shown that mitochondrial β-oxidation of long-chain fatty acids increases the ratio between electrons entering the respiratory chain via FADH2 or NADH (the longer the fatty acid, the higher the ratio), leading to higher levels of reactive oxygen species (ROS) generation, thereby impairing mitochondrial function. Consistent with the increased response to oxidative stress in colonocytes (Fig. S6c), mitochondrial function was impaired in the colon tissues of HFD mice, with decreased mitochondrial gene expression (Fig. 5b) and oxidative phosphorylation (Fig. 5c) as well as reduced ATP levels (Fig. up to this point.

In colonocytes, oxidative phosphorylation of mitochondria consumes oxygen to maintain hypoxia in the colonic epithelium, which maintains the anaerobic nature of the colonic lumen, promoting the dominance of obligate anaerobes while suppressing facultative species such as Enterobacteriaceae growth of anaerobic bacteria. To investigate the oxygenation of the colonic epithelium in response to impaired mitochondrial bioenergetics on HFD, we used the exogenous hypoxia marker pimonidazole to observe the hypoxia of the colonic epithelium (Fig. 5d). Pimonidazole staining revealed that colonic epithelial hypoxia disappeared in HFD-treated mice (Fig. 5e), suggesting increased availability of luminal oxygen, a major factor regulating the growth of the Proteobacteria phylum in the gut. Increased oxygen concentrations disrupted the physiological hypoxia of colonocytes (Fig. 5g), which is critical for the adaptation of cellular metabolism and various processes such as barrier function and immunity. An important immune feature of the colon is the highly glycosylated and hydrated mucus layer, which provides a source of nutrition for gut microbiota such as Akkermansia. Consistent with increased epithelial oxygenation, sustained HFD suppressed colonic mucus production, as revealed by immunofluorescence of intestinal secreted mucin-2 (MUC2) (Fig. S7a).

Impaired mitochondrial bioenergetics led to a shift in colonocyte metabolism to glycolytic metabolism, even under aerobic conditions (Fig. 5h), characterized by high lactate release, low oxygen consumption, and increased nitrate synthesis. To investigate the reprogramming of colonic metabolism in HFD mice, an untargeted metabolomics study was performed. PCA results showed that the metabolic profiles of Chow and HFD mice were quite different (P = 0.006) (Fig. 5i). Consistent with the results of colonic RNA-sequencing (Fig. 4b, 5g,h and S6a-b), qMSEA revealed that HFD-altered colonic metabolites (n = 554, P < 0.05) were involved in fatty acid metabolism, β-oxidation of very long chain fatty acids , Trp metabolism and the hallmark phenomenon of aerobic glycolysis, the Warburg effect (Fig. 5j). At the same time, we detected a significant increase in lactate concentration (P < 0.01) in the colonic contents of HFD mice (Fig. 5k) and a significant increase in nitrate concentration in the colon tissue (P < 0.001) (Fig. 5l). Host-derived nitrate can be used by several bacteria such as Enterobacteriaceae as electron acceptors for anaerobic respiration to produce ATP. The redox reactions in which bacteria exploit the greatest free energy predominate, which determines which metabolic bacterial populations can dominate a habitat's microbial community. These results suggest that HFD-induced mitochondrial damage and subsequent reorientation of colonocyte metabolism increases the accessibility of respiratory substrates of the Proteobacteria as a potential mechanism for HFD-induced gut dysbiosis.

Figure 5 HFD disrupts mitochondrial function and causes metabolic reorientation in colonocytes. a HFD decreased butyrate concentrations in colonic contents (n = 6 per group). HFD inhibited mitochondrial gene expression (b) and mitochondrial oxidative phosphorylation (c). e Quantification of pimonidazole staining by blind scoring of colon sections (3 mice, n = 9). f Adenosine triphosphate (ATP) concentrations in colon tissue (n = 8 per group). g HFD downregulates HIF-α-mediated epithelial hypoxia in colon tissue. hHFD induces a shift in short-chain fatty acid (SCFAs) metabolism to glycolysis in colonic tissue. iPCA was used to evaluate the comparison of colonic metabolome data from HFD mice to Chow mice (n = 6 per group). j Colonic metabolic pathways disturbed by HFD were identified using the qMSEA method and the top 30 pathways are shown. HFD increased lactate in colonic contents (k, n = 6) and nitrate in colonic tissue (l, n = 8).

⑥Remodeling colon mitochondrial function

Alleviate Trp-Kyn metabolic disorder caused by intestinal flora imbalance

Remodeling of colonic mitochondrial bioenergetics alleviated the gut dysbiosis-induced dysregulation of Trp-Kyn metabolism

To confirm a causal relationship between colonocyte mitochondrial dysfunction and Proteobacteria overgrowth, standard diet-fed mice were treated with the PPAR-γ antagonist GW9662 to inhibit colonocyte mitochondrial oxidative phosphorylation (C-GW) (Fig. 6a ). Consistent with the observations in HFD mice, C-GW mice had decreased ATP concentrations (Fig. 6c) and increased colonic tissue epithelial oxygenation (Fig. 6b) and nitrate production (Fig. 6d). Furthermore, 16S rRNA analysis revealed that inhibition of colonic mitochondrial bioenergetics by GW9662 altered gut bacterial composition (Fig. 6e), characterized by increased abundance of Proteobacteria and overgrowth of Desulfovibrio and Enterobacteriaceae (Fig. 6f ,g). Meanwhile, colonic IDO1 upregulation (Fig. 6h) and serum metabolic disturbances characterized by enhanced Kyn pathway (Fig. 6i,j) were also observed in C-GW mice, suggesting that colonic mitochondrial dysfunction plays an important role in gut microbiota dysregulation. and subsequent Trp-Kyn metabolic dysregulation.

Given that bacterially fermented butyrate is reduced in colonic contents (Fig. 5a), leading to a shift in colonocyte energy metabolism towards β-oxidation of long-chain and very long-chain fatty acids (Fig. 5j and S6a-b), we wanted to investigate Whether salt remodeling mitochondrial bioenergetics in colon cells can alleviate HFD-induced Trp-Kyn metabolic dysregulation. To this end, HFD mice were fed with butyrate in their drinking water for 4 weeks (H-Buy) (Fig. 6a). In HFD mice, butyrate supplementation restored colonic epithelial cell hypoxia (Fig. 6b) and mitochondrial activity (Fig. 6c), and reduced nitrate production in colonic tissues (Fig. 6d). Consistent with a reduction in the utilization of respiratory electron acceptors by the Proteobacteria phylum, H-Buy mice displayed different bacterial profiles compared to HFD mice (Fig. 6e,f), especially a reduction in the number of LPS-producing bacteria (Fig. 6g). , suggesting that butyrate has a beneficial effect on the gut microbiota. Furthermore, compared with HFD mice, H-Buy mice had lower colonic IDO1 expression (Fig. 6h) and downregulated Kyn pathway (Fig. 6j). Taken together, these results re-emphasize the criticality of the interplay between gut microbiota and colonocytes for maintaining systemic Trp-Kyn metabolic balance.

Figure 6. Gut microbiota dysbiosis caused by colonic mitochondrial dysfunction disrupts systemic Trp-Kyn metabolism. a GW9662 was administered to mice fed a standard diet to study the relationship between colonic mitochondrial dysfunction and Trp metabolism (C-GW). To demonstrate the beneficial effects of remodeling colonic mitochondrial bioenergetics on systemic Trp-Kyn metabolism, mice fed a high-fat diet were provided with butyrate in their drinking water (H-Buy). b Oxygenation of colonic epithelial cells was measured by pimonidazole (red). Nuclei were counterstained with DAPI (blue) (9 sections from 3 mice). Concentrations of ATP (c) and nitrate (d) in colon tissue (n = 8 per group). e PCoA showing similarity to gut microbiota as determined by 16S sequencing. f Relative abundance of bacterial phyla. h Fluorescent immunostaining for IDO1 (green) in mouse colon sections. iPCA shows serum metabolic profile. j Serum metabolites involved in the Kyn pathway (n = 6 per group).

- discuss -

A cross-sectional study showed that a Western-style diet was accompanied by abnormal Trp metabolism characterized by upregulation of the Kyn pathway and depletion of Trp. Recent evidence suggests that overexpressed IDO1 in adipocytes is thought to be responsible for excess Kyn in rodent models of HFD. Likewise, most previous studies have focused on tissues that specifically express IDO1, such as liver, muscle, and adipocytes, whereas the role of the gut microbiota in diet-associated Trp metabolic disorders has been less studied. Accumulating evidence indicates that the gut microbiota is a central mediator of diet-related chronic diseases and metabolic syndrome. In this study, we demonstrated that HFD impairs colonocyte mitochondrial bioenergetics, promotes the expansion of Proteobacteria, and then upregulates colonocyte inflammatory response and subsequent IDO1 expression, finally leading to systemic Trp-Kyn metabolic disturbance. Below, we discuss how these findings broaden our understanding of the central role and underlying mechanisms of gut microbiota-colonocyte interactions in diet-disturbed Trp metabolism.

Generally, less than 1% of Trp obtained through diet is used for protein synthesis, and more than 95% is metabolized through the Kyn pathway. The tissue-specifically expressed rate-limiting enzymes in the Kyn pathway are IDO1 and tryptophan-2,3-dioxygenase (TDO), the two most well-studied enzymes. TDO is not expressed in the gut and will not be discussed here. In addition, the expression pattern of IDO1 is more relevant to pathology. For example, colon tissues from patients with inflammatory bowel disease and colorectal cancer are often accompanied by high expression of IDO1. In some cases, Kyn is considered an immunosuppressant, partly due to the concomitant increase of Kyn following inflammation. However, the researchers also found that injecting exogenous Kyn into mice did not alleviate the inflammatory microenvironment and, on the contrary, exacerbated insulin resistance, suggesting that increased circulating Kyn negatively impacts individual health. These findings suggest that Kyn and its metabolites play complex roles in various pathological conditions. However, the association of Trp-Kyn pathway abnormalities with human disease is widely accepted. The CNS uptakes approximately 60% of Kyn from circulation; thus, hyperfunction of peripheral Kyn pathways, often triggered by inflammation, may initiate or exacerbate CNS disorders. Kyn is usually hydroxylated to 3-HK and then further converted to quinones, resulting in neurotoxicity. Although the negative impact of abnormal Kyn metabolism on human health is well established, the source of abnormal increase in peripheral Kyn has remained elusive.

Increased consumption of Western-style diets, including ultra-processed foods and convenience products, has been linked to the development of non-communicable diseases, which are responsible for more than 80% of deaths in Westernized societies today. Long-term Western-style diet can disrupt physiological balance through pathological changes in blood lipids, induction of metabolic syndrome, and overactivation of the immune system. More and more studies have shown that tissue-specific and systemic immune responses are highly integrated with metabolic regulation, which is considered to be the core of maintaining body balance. Low-grade chronic inflammation is thought to contribute to IDO1 activation. Meanwhile, hyperactivation of the Kyn pathway is also associated with pathological development under inflammatory conditions. There is increasing evidence that diet-related diseases are associated with dysbiosis of the gut microbiota. Mainly located in the human colon, the commensal gut microbiota is a variable and complex system that requires continuous barriers and regulatory mechanisms to maintain host-microbe interactions, tissue and immune balance, and the overall physiology of the individual. Among the various factors affecting the gut microbiota, food intake and dietary habits have a major impact on the composition and function of the microbiota. Sustained high-density lipoprotein diets in mice resulted in dysbiosis of the gut microbiota, with an overgrowth of pathogenic bacteria (Proteobacteria) and a decrease in beneficial bacteria to the host (Fig. 2c–f). The development of techniques to study microbe-host interactions, such as germ-free mice and FMT, has provided us with the tools to study the causal role of the gut microbiota in disease progression. Trp-Kyn metabolism was disturbed in standard diet-fed mice receiving FMT from HFD mice (Fig. 3g,h), whereas Trp-Kyn metabolism was abolished in HFD mice treated with antibiotic cocktails (Fig. 3i). These results demonstrate for the first time the central role of gut microbiota in HFD-induced dysregulation of Trp-Kyn metabolism. Mechanistically, a dysbiotic microbiota stimulates the intestinal mucosal immune system through several mechanisms, such as altered signaling through Toll-like receptors (TLRs) (Fig. 4d,e), while reducing mucus release into the lumen ( Figure S7a). These factors in turn lead to disruption of barrier integrity and dysregulation of intestinal immune balance, leading to upregulation of colonic IDO1 expression and increased peripheral Kyn concentrations (Fig. 4b,c and 7b).

Animal models have demonstrated that the metabolism of colonocytes acts as a control switch, bridging the gap between balance and dysbiosis of the gut microbiome (Fig. 7a). Accumulating evidence suggests that HFD disrupts host control of the microbiome, leading to structural shifts in gut commensal flora. Our results suggest that sustained HFD perturbs the Bacteroides/Firmicutes ratio (Fig. 2d), a signature of the gut microbiota in obese patients. Colonic anaerobic bacteria convert undigested dietary fiber into fermentation products, which are directly absorbed by colonocytes and then oxidized in mitochondria, leading to increased oxygen consumption of epithelial cells and placing epithelial cells in a physiological hypoxic state (Fig. 5d). Among the metabolites of bacterial fermentation, butyrate is the main energy source (over 70%) for colonocytes and activates PPAR-γ, enhancing mitochondrial β-oxidation and oxygen consumption. Low levels of intracellular oxygen concentration inhibit prolyl hydroxylase and HIF-inhibiting factors to maintain HIF-1α and translocate it to the nucleus, which is related to the regulation of intestinal epithelial metabolism and barrier function (Fig. 5j and S7a ). Furthermore, hypoxia in the epithelium limits the diffusion of oxygen to the lumen of the colon, which supports an anaerobic environment in the lumen of the colon and promotes the dominance of beneficial anaerobic bacteria in the gut microbiota (Fig. 7a). These insights point to a critical role of mitochondrial bioenergy in shaping the gut microbiota by colonocyte metabolism.

In the present study, we noticed that sustained high-frequency breakdown increased β-oxidation of long-chain and very long-chain fatty acids in colonocytes (Fig. 5j and S6a–b). Oxygen radical formation is accompanied by β-oxidation in mitochondria and is determined by the substrates of mitochondrial respiration. Dave proposed a kinetic model in which the ratio between electrons entering the respiratory chain via FADH2 or NADH (the F/N ratio) is a key determinant of ROS formation in mitochondria. During glucose oxidation, this ratio is low (0.2), while the F/N ratio (15/31) of long chain fatty acids (16 C atoms) such as palmitic acid is 0.48. Oxidative stress caused by ROS accumulation impairs mitochondrial bioenergetics in colonic epithelial cells (Fig. 5b,c,f). Since mitochondrial oxygen consumption maintains physiological hypoxia on the colonic surface, sustained HFD impairs mitochondrial function to increase colonic epithelial oxygenation (Fig. 5d), thereby increasing the oxygen supply to the colonic lumen and promoting Expansion of Proteobacteria. At the same time, decreased mitochondrial activity in colonic epithelial cells induced the release of nitrate (Fig. 5l), which also serves as an electron acceptor for Proteus. Notably, sustained HFD enriched the colon for Desulfovibrio (Fig. 2e,f), which normally grows under anaerobic conditions and can tolerate low levels of air exposure. An important feature of Desulfovibrio is its ability to use lactate as an electron donor for respiration. Impaired mitochondrial function in the colon led to a shift in colonocytes from oxidative phosphorylation to anaerobic glycolysis, which was characterized by low oxygen consumption and increased lactate production and secretion (Figures 5k and 7b), supporting the expansion of Desulfovibrio. In addition to producing LPS, Desulfovibrio can also use sulfate to produce hydrogen sulfide. Interestingly, HFD mice were also enriched in other hydrogen sulfide producers, including Streptococcus and Haemophilus bisporum (Fig. 2f). High levels of hydrogen sulfide have been reported to be cytotoxic and may cause inflammation of the colon. The causal relationship between impaired mitochondrial bioenergetics in colonocytes and HFD-induced dysregulation of peripheral Trp-Kyn metabolism was further confirmed by inhibiting colonic mitochondrial β-oxidation in standard diet-fed mice using the PPAR-γ antagonist GW9662 (Fig. 6).

Antibiotic treatment depletes the butyrate-producing gut bacterial population, thereby disrupting the metabolism of the colonic epithelium, and studies of the consequences of antibiotic treatment provide an initial understanding of the mechanisms that destabilize the gut microbiota. Given the maintenance role of butyrate in colonic metabolism, we propose that a shift in colonocyte mitochondrial respiration to long-chain fatty acids may be responsible for the decrease in colonic butyrate. At the same time, the researchers found that by feeding antibiotic-treated mice a diet rich in plant fiber or activating the downstream pathway of butyrate (that is, using a PPAR-γ agonist, such as 5-aminosalicylic acid), the Colon resurfacing in physiological hypoxia. Consistent with previous studies, butyrate supplementation in drinking water significantly restored the hypoxic state of the colonic epithelium and reduced nitrate production, thereby reducing the abundance of Proteobacteria, which contributed to the suppression of colonic IDO1 expression and peripheral Trp-Kyn metabolism (Figure 6).

Fig. 7 Schematic diagram of the mechanism of HFD leading to peripheral Trp-Kyn metabolic disorder. a In a healthy gut, the gut microbiota is dominated by obligate anaerobic bacteria that convert fiber into fermentation products such as butyrate to maintain colonocyte metabolism. b Sustained HFD reduces colonic butyrate concentrations and enhances β-oxidation of long-chain and very long-chain fatty acids in colonocytes, thereby impairing mitochondrial bioenergetics, leading to metabolic reprogramming of colonocytes, and enabling Proteobacteria to proliferate . Bacterial-derived LPS stimulates colonic immune responses and upregulates the IDO1-mediated Kyn pathway.

references:

Sun, P., Wang, M., Liu, YX. et al. High-fat diet-disturbed gut microbiota-colonocyte interactions contribute to dysregulating peripheral tryptophan-kynurenine metabolism. Microbiome 11, 154 (2023). https://doi.org/10.1186/s40168-023-01606-x

- First author -

NWAFU

School of Veterinary Medicine

Sun Penghao

PhD candidate

The first author: Sun Penghao, a doctoral student at the School of Veterinary Medicine, Northwest A&F University, mainly focuses on the impact of the interaction between diet and intestinal flora on the health of the host. At present, he has published related papers in Microbiome, Theranostics, Journal of Affective Disorders and Stress Biology as the first author and co-first author.

NWAFU

School of Veterinary Medicine

Wang Mengli

PhD candidate

Co-first author: Wang Mengli, a doctoral student at the School of Veterinary Medicine, Northwest A&F University, mainly focuses on the improvement of neurological diseases by regulating the host symbiotic flora with prebiotics. At present, he has published related papers in Microbiome, Theranostics, Journal of Affective Disorders and Stress Biology as the first author and co-first author.

- Corresponding Author -

NWAFU

Zhao Shanting

Zhao Shanting, male, born in September 1964 in Gaoqing County, Shandong Province, German Medical Doctor, Distinguished Professor of "Houji Scholar" of Northwest A&F University, Doctoral Supervisor, Librarian of Northwest A&F University, "Innovative Talent of Shaanxi Province" Distinguished expert for long-term projects, the first batch of "Tianfu Scholars" distinguished experts in Sichuan Province, deputy director of Yuanbaofeng Engineering Technology Research Center of State Forestry and Grassland Administration. Now concurrently serves as vice chairman of Overseas Chinese Federation of Northwest A&F University, deputy director of Yuanbaofeng Scientific Research Industrialization Development Research Center, deputy director of Eucommia Research Institute, deputy director of Sino-US Food Safety Center, deputy director of Shaanxi Provincial Enzyme Standards Committee, etc. He used to be an assistant professor at the University of Freiburg in Germany, a researcher at the Center for Molecular Neuroscience (ZMNH) at the University of Hamburg, and a distinguished professor of "Cuiying Scholars" at Lanzhou University. He is currently a member of the CPPCC Yangling District, an executive director of the Chinese Society for Animal Welfare and Healthy Breeding, an executive director of the Chinese Animal Anatomy and Histology and Embryology, a member of the American Academy of Neuroscience, a member of the European Academy of Neuroscience, a member of the German Anatomical Society, and a member of the Chinese Academy of Neuroscience. Member of Chinese Anatomy Society, executive director of Shaanxi Natural Medicine Society. Reviewer of internationally renowned academic journals of developmental biology and neurobiology "Development", "Journal of Neuroscience", "European Journal of Neuroscience", "Journal of Comparative Neurology", "Neuroscience".

NWAFU

Zhu Xiaoyan

Zhu Xiaoyan, female, native of Duolun, Inner Mongolia, member of the Communist Party of China, doctor, associate professor, doctoral supervisor. Member of Animal Anatomy and Histology and Embryology Branch of Chinese Animal Husbandry and Veterinary Society, Chinese Neuroscience Society, American Neuroscience Society, American Society of Animal Science and Shaanxi Natural Medicine Society, and member of the Biochemistry and Molecular Pharmacology Committee of Shaanxi Pharmacology Society. Communication review expert of the Life Sciences Department of the National Natural Science Foundation of China, and communication review expert for dissertations of the Ministry of Education. Reviewer of "Journal of Neuroinflammation", "Journal of Neurochemistry", "Frontiers in Pharmacology" and other journals.

Xi'an Medical College

Chai Xuejun

Chai Xuejun, female, born in July 1968, German-Chinese, winner of the German "Martini Medical Talent Award", a special expert of Sichuan Guangyuan Haizhi Work Base, a candidate for the long-term innovation project of high-level talents in Shaanxi Province, and a special expert of Shaanxi Province , Professor of Human Anatomy and Histology and Embryology, Xi'an Medical College. The academic backbone of the key discipline construction project of Xi'an Medical College. Bachelor of Clinical Medicine, Xinjiang Medical University, Master of Obstetrics and Gynecology, Doctor of Neurobiology at the University of Freiburg, Germany, postdoctoral fellow, senior researcher at the University of Hamburg, Germany, and leader of the research group. Hosted and participated in 7 scientific research projects of the German Research Foundation (DFG). After returning to China, he was approved for one major project of the Shaanxi Provincial Major Guidance Plan, one general project of the Shaanxi Provincial Natural Science Basic Research Plan, one project for the Revitalization of Talents of Xi'an Medical College, and several innovation funds for college students. Published a total of 37 academic papers, including 10 as the first author and 4 corresponding authors, among which they were published in internationally renowned academic journals of neurobiology and developmental biology such as "Nature Protocols", "J Neurosci", "Cerebral Cortex" and "Development" He has published 30 SCI papers, more than half of which are in the first and second districts of the Chinese Academy of Sciences, with a cumulative impact factor of more than 120 points. He has a high reputation and influence in the research on the mechanism of brain development and related diseases. He has been invited to participate in domestic and foreign academic conferences and give conference reports. Guest editor of "Frontiers in Neuroenergetics, Nutrition and Brain Health", reviewer of "Cerebral Cortex" and "Development", member of American Academy of Neuroscience, member of European Academy of Neuroscience, member of German Anatomical Society, member of Chinese Academy of Neuroscience, Shaanxi Member of the Professional Committee of the Provincial Pharmacological Society, member of the Shaanxi Provincial Brain Disease Molecular Medicine Professional Committee, and Deputy Secretary-General of the First Council of the Shaanxi Natural Medicine Society.

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Microbiome | Wesfarmers reveals the mechanism by which high-fat diet disrupts peripheral tryptophan-kynurenine metabolic homeostasis

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