Multiomics Analysis Reveals Leucine Deprivation Promotes Bile Acid Synthesis by Upregulating Hepatic CYP7A1 and Intestinal Turicibacter sanguinis in Mice.

BACKGROUND: Leucine, a branched-chain amino acid, participates in the regulation of lipid metabolism and the composition of the intestinal microbiota. However, the related mechanism remains unclear. OBJECTIVES: Here, we aimed to reveal the potential mechanisms by which hepatic CYP7A1 (a rate-limiting enzyme for bile acid [BA] synthesis) and gut microbiota coregulate BA synthesis under leucine deprivation. METHODS: To this end, 8-wk-old C57BL/6J mice were fed with either regular diets or leucine-free diets for 1 wk. Then, we investigated whether secondary BAs were synthesized by Turicibacter sanguinis in 7-wk-old C57BL/6J germ-free mice gavaged with T. sanguinis for 2 wk by determining BA concentrations in the plasma, liver, and cecum contents using liquid chromatography-tandem mass spectrometry. RESULTS: The results showed that leucine deprivation resulted in a significant increase in total BA concentration in the plasma and an increase in the liver, but no difference in total BA was observed in the cecum contents before and after leucine deprivation. Furthermore, leucine deprivation significantly altered BA profiles such as taurocholic acid and ω-muricholic acid in the plasma, liver, and cecum contents. CYP7A1 expression was significantly upregulated in the liver under leucine deprivation. Leucine deprivation also regulated the composition of the gut microbiota; specifically, it significantly upregulated the relative abundance of T. sanguinis, thus enhancing the conversion of primary BAs into secondary BAs by intestinal T. sanguinis in mice. CONCLUSIONS: Overall, leucine deprivation regulated BA profiles in enterohepatic circulation by upregulating hepatic CYP7A1 expression and increasing intestinal T. sanguinis abundance. Our findings reveal the contribution of gut microbiota to BA metabolism under dietary leucine deprivation.

Multi-Omics Analysis Reveals Sphingomyelin Accumulation, Glycerolipids Loss, and Disorders of Lipid Metabolism Regulated by Leucine Deprivation in the Liver of Mice.

SCOPE: Branched-chain amino acids, especially leucine, have been reported to play a role in regulating lipid metabolism. This study aims to examine the effects of leucine deprivation on hepatic lipid metabolism. METHODS AND RESULTS: C57BL/6 mice are fed with a chow diet (control group, n = 8) or a leucine-free diet (-Leu group, n = 8) for 7 days. Histology, lipidomics, targeted metabolomics, and transcriptomics are performed to analyze the liver tissue. Compared to control group, -Leu group exhibits a notably reduced liver weight, accompanied by hepatic injury, and disorders of lipid metabolism. The level of sphingomyelin (SM) is significantly increased in the liver of -Leu group, while the glycerolipids (GL) level is significantly decreased. The expression of sphingomyelin synthase 1 (SGMS1) is upregulated by leucine deprivation in a time-dependent manner, leading to hepatic SM accumulation. Moreover, leucine deprivation results in hepatic GL loss via suppressing fatty acid synthase (FASN) and acetyl-CoA carboxylase 1 (ACC1) expression. CONCLUSION: The findings demonstrate that leucine deprivation results in abnormal lipid metabolism in the liver, mainly manifested as SM accumulation and GL loss. These results provide insights into the role of leucine in regulating lipid metabolism.

Lysine crotonylation regulates leucine-deprivation-induced autophagy by a 14-3-3ε-PPM1B axis.

Lysine crotonylation as a protein post-translational modification regulates diverse cellular processes and functions. However, the role of crotonylation in nutrient signaling pathways remains unclear. Here, we find a positive correlation between global crotonylation levels and leucine-deprivation-induced autophagy. Crotonylome profiling identifies many crotonylated proteins regulated by leucine deprivation. Bioinformatics analysis dominates 14-3-3 proteins in leucine-mediated crotonylome. Expression of 14-3-3ε crotonylation-deficient mutant significantly inhibits leucine-deprivation-induced autophagy. Molecular dynamics analysis shows that crotonylation increases molecular instability and disrupts the 14-3-3ε amphipathic pocket through which 14-3-3ε interacts with binding partners. Leucine-deprivation-induced 14-3-3ε crotonylation leads to the release of protein phosphatase 1B (PPM1B) from 14-3-3ε interaction. Active PPM1B dephosphorylates ULK1 and subsequently initiates autophagy. We further find that 14-3-3ε crotonylation is regulated by HDAC7. Taken together, our findings demonstrate that the 14-3-3ε-PPM1B axis regulated by crotonylation may play a vital role in leucine-deprivation-induced autophagy.

Multi-omics analysis reveals gut microbiota-induced intramuscular fat deposition via regulating expression of lipogenesis-associated genes.

The gut microbiome has great effects on the digestion, absorption, and metabolism of lipids. However, the microbiota composition that can alter the fat deposition and the meat quality of pigs remains unclear. Here, we used Laiwu (LW) pigs (a native Chinese breed with higher intramuscular fat) compared with commercial crossbreed Duroc × (Landrace × Yorkshire) (DLY) pigs to investigate the effects of microbiota on meat quality, especially in intramuscular fat content. A total of 32 DLY piglets were randomly allotted to 4 groups and transplanted with fecal microbiota from healthy LW pigs. The results indicated that the high dose of fecal microbiota transplantation (HFMT) selectively enhanced fat deposition in longissimus dorsi (P < 0.05) but decreased backfat thickness (P < 0.05) compared with control group. HFMT significantly altered meat color and increased feed conversation ratio (P < 0.05). Furthermore, the multi-omics analysis revealed that Bacteroides uniformis, Sphaerochaeta globosa, Hydrogenoanaerobacterium saccharovorans, and Pyramidobacter piscolens are the core species which can regulate lipid deposition. A total of 140 male SPF C57BL/6j mice were randomly allotted into 7 groups and administrated with these 4 microbes alone or consortium to validate the relationships between microbiota and lipid deposition. Inoculating the bacterial consortium into mice increased intramuscular fat content (P < 0.05) compared with control mice. Increased expressions of lipogenesis-associated genes including cluster of differentiation 36 (Cd36), diacylglycerol O-acyltransferase 2 (Dgat2), and fatty acid synthase (FASN) were observed in skeletal muscle in the mice with mixed bacteria compared with control mice. Together, our results suggest that the gut microbiota may play an important role in regulating the lipid deposition in the muscle of pigs and mice.

Integrated analysis of multi-tissues lipidome and gut microbiome reveals microbiota-induced shifts on lipid metabolism in pigs.

Lipid metabolism is very important for meat quality in pigs. Accumulating evidence shows that gut microbiota can contribute to this physiological process. However, the gut microbiota that function in lipid metabolism and adipogenesis remains unclear. Here, we compared the characteristics of fat deposition and gut microbial community between Laiwu pigs and Duroc × (Landrace × Yorkshire) (DLY) pigs. Fecal microbiota transplantation (FMT) was performed to determine the possible impact of gut microbiota on lipid metabolism in pigs. An integrated analysis of the gut microbiome and lipidome of the small intestine, plasma, and liver was conducted to investigate the effects of FMT on host lipid metabolism. The comparative analysis of the gut microbiome showed higher abundance of Bacteroidetes (P = 0.0018) while lower abundance of Firmicutes (P = 0.012) in Laiwu pigs, and the microbial composition can be transferred from Laiwu pigs into DLY pigs. Transmission electron microscope and Oil red-O staining were performed to analyze the effects of FMT on lipid deposition in liver, the main target organ for lipid metabolism. The results showed that FMT significantly increased the number of lipid droplets (P = 0.0035) and lipid accumulation (P = 0.0026) in liver. Furthermore, integrated multi-tissues lipidome analysis demonstrated that the fatty acyls and glycerophospholipids were significantly increased (P < 0.01) in intestine and liver, while glycerolipids and fatty acyls were reduced (P < 0.01) in plasma. In the small intestine, FMT increased (P < 0.01) the relative abundance of polyketides and prenol lipids but reduced (P < 0.01) the saccharolipids. Correlation analysis revealed the potential interactions between microbiota and lipid metabolites. Together, our results indicated that the gut microbiota may regulate the lipid metabolism and enhance the accumulation of lipid droplets in the liver of pigs.

Dietary ε-Polylysine Affects on Gut Microbiota and Plasma Metabolites Profiling in Mice.

Given the antibacterial effects of ε-polylysine acting on cell membranes, and that glycerol phospholipids are important components of the cell membrane, we hypothesized that ε-polylysine may regulate glycerophospholipid metabolism by modifying the gut microbiota. To test this hypothesis, we treated post-weaning C57 mice with different levels of ε-polylysine (0, 300, 600, and 1,200 ppm) in their basic diet. The growth performance and morphology of intestine were then determined. Modification of the gut microbiota and their function were analyzed using 16S rDNA sequencing. Metabolite identification was performed using the LC-MS method. The results showed that body weight decreased with an increasing supplemental level of ε-polylysine from 5 to 7 weeks (P < 0.05), but no significant difference was observed after 8 weeks (P > 0.05). Supplementation with 1,200 ppm ε-polylysine changed the morphology of the jejunum and ileum, increased the villus length, decreased the crypt depth of the jejunum, and decreased the villus length and crypt depth of the ileum (P < 0.05). ε-Polylysine shifted the intestine microbiota by changing alpha diversity (Chao 1, observed species, Shannon, and Simpson indices) and varied at different times. ε-polylysine decreased Firmicutes and increased Bacteroidetes at 4 week, but increased Firmicutes and decreased Bacteroidetes at 10 week. ε-Polylysine regulated genera associated with lipid metabolism such as Parabacteroides, Odoribacter, Akkermansia, Alistipes, Lachnospiraceae UCG-001, Collinsella, Ruminococcaceae, and Intestinimonas. During the adult period, the genera Alistipes, Lachnospiraceae UCG-001, and Streptomyces were positively associated with PC, PE, LysoPC, LysoPE, 1-Arachidonoylglycerophosphoinositol and OHOHA-PS (R > 0.6, P < 0.001), but changes in Blautia, Christensenellaceae R-7 group, Odoribacter, Allobaculum, Ruminococcaceae UCG-004, Ruminococcaceae UCG-005, and Lachnospiraceae UCG-010 were negatively correlated with glycerophospholipid metabolites (R < -0.6, P < 0.001). The abundance of glycerophospholipid metabolites, including PC, PE, lysoPC, and lysoPE, were decreased by ε-polylysine. Furthermore, ε-polylysine reduced the incidence of the genera including Ruminococcus, Prevotella, Prevotellaceae, Butyricimonas, and Escherichia-Shigella and reduced the abundance of Faecalibaculum, Christensenellaceae R-7 group, Coriobacteriaceae UCG-002. In conclusion, ε-polylysine modified gut microbiota composition and function while also restraining pathogenic bacteria. The glycerophospholipid metabolism pathway and associated metabolites may be regulated by intestinal bacteria.

Dietary glycyl-glutamine supplementation ameliorates intestinal integrity, inflammatory response, and oxidative status in association with the gut microbiota in LPS-challenged piglets.

During weaning transition, mammalian newborns suffer severe enteric infections and thus induced gut microbiota dysbiosis, which in turn aggravates enteric disorder. The synthetic dipeptide glycyl-glutamine (GlyGln) has been used as a diet supplement to improve the weaning transition of newborns. However, the effect of dietary GlyGln supplementation on the gut microbiota of piglets with enteric infection remains unclear. Here, weaned piglets received a basal diet or a basal diet supplemented with 0.25% GlyGln for 3 weeks. Five piglets in each group received an intraperitoneal injection of lipopolysaccharide (LPS) (100 µg per kg BW) (LPS and GlyGln + LPS groups) and meanwhile five piglets in a control group received an intraperitoneal injection of saline (Ctrl group). The results showed that dietary GlyGln supplementation improved the LPS induced inflammation response and damage to the ileum morphology by increasing interleukin 10, tight junction proteins, villus height, and the ratio villus height/crypt depth, but decreasing the crypt depth. For the oxidative status, dietary GlyGln supplementation increased the ileal superoxide dismutase and meanwhile reduced the malondialdehyde and nitric oxide synthase activity (NOS) (total NOS and inducible NOS), compared with that in the LPS group. LPS challenge reduced the diversity of gut microbiota and enriched the facultative anaerobic Escherichia coli. The GlyGln restored alpha diversity and the structure of the gut microbiota by enriching obligate anaerobes and short-chain fatty acid (SCFA)-producing bacteria, including Clostridium, Lachnospira, Phascolarctobacterium, Roseburia, Lachnospiraceae, and Synergistetes. GlyGln enriched the gut microbiota function of carbohydrate metabolism and elevated the ileal SCFA concentrations of propionic acid and butyric acid that had been decreased by the LPS challenge. The beneficial effects of dietary GlyGln supplementation are closely associated with its enriched bacteria and SCFAs. Taken together, dietary GlyGln supplementation improved the gut microbiota dysbiosis induced by LPS challenge and enriched obligate anaerobes and SCFA-producing bacteria, which contributed to the amelioration of intestinal integrity, inflammatory responses, and oxidative status.

Modulation of Gut Microbial Community and Metabolism by Dietary Glycyl-Glutamine Supplementation May Favor Weaning Transition in Piglets.

Gut microbiota plays a crucial role in diet nutrient metabolism and maintaining host health. The synthetic dipeptides glycyl-glutamine (Gly-Gln) used as diet supplementation to improve the weaning transition of newborns could be metabolized by certain bacteria in vitro. However, the effect of diet Gly-Gln supplementation on gut microbiota in vivo remains largely unknown. 240 piglets at the age of 28 days (day 28) were randomly assigned to two groups that received a basal diet (Ctrl group) or a basal diet supplemented with 0.25% Gly-Gln (Gly-Gln group) for 3 weeks. Five piglets from each group were euthanized for sampling after overnight fasting on day 38 and day 49, respectively. We determined their structure shifts of the gut microbiota using 16S rDNA-based high-throughput sequencing analysis. Microbial metabolites short-chain fatty acids (SCFAs) in the ileum and the colon were determined with high-performance gas chromatography. The concentrations of endocrine peptides including epidermal growth factor, glucagon-like peptide-1, and glucagon-like peptide-2 in ileal mucosa, as well as the serum concentration of interleukin 1 beta, interleukin 6, interleukin 10, and tumor necrosis factor alpha were determined using Enzyme-Linked Immunosorbent Assay. In addition, we also checked the diarrhea ratio, growth performance, and intestinal morphology to assess the favorable effect of dietary Gly-Gln supplementation during the weaning transition. Dietary Gly-Gln supplementation beneficially altered the gut microbiota by increasing bacterial loading, elevating alpha diversity, and increasing the relative abundance of anaerobes and fiber-degrading bacteria (Phylum Fibrobacteres). Accordingly, the microbial metabolites SCFAs in both colon and ileum, as well as the downstream endocrine peptides in the ileum increased. Meanwhile, dietary Gly-Gln's favorable weaning transition was reflected in the increase of growth performance indices and the reduced inflammatory response in a time dependent manner. There were significant correlations among the bacteria which responded to dietary Gly-Gln supplementation and these checked indices. Taken together, dietary Gly-Gln supplementation selectively modulated the gut microbiota, which may favor piglets' weaning-transition. These findings suggest that gut microbiota targeted approaches can be potentially used to improve weaning transition of piglets by dietary functional amino acid.

A Microbiota-Derived Bacteriocin Targets the Host to Confer Diarrhea Resistance in Early-Weaned Piglets.

Alternatives to antibiotics for preventing diarrhea in early-weaned farm animals are sorely needed. CM piglets (a native Chinese breed) are more resistant to early-weaning stress-induced diarrhea than the commercial crossbred LY piglets. Transferring fecal microbiota, but not saline, from healthy CM into LY piglets by oral administration prior to early weaning conferred diarrhea resistance. By comparing the relative abundance of intestinal microbiota in saline and microbiota transferred LY piglets, we identified and validated Lactobacillus gasseri LA39 and Lactobacillus frumenti as two bacterial species that mediate diarrhea resistance. Diarrhea resistance depended on the bacterial secretory circular peptide gassericin A, a bacteriocin. The binding of gassericin A to Keratin 19 (KRT19) on the plasma membrane of intestinal epithelial cells was essential for enhancement of fluid absorption and decreased secretion. These findings suggest the use of L. gasseri LA39 and L. frumenti as antibiotic alternatives for preventing diarrhea in mammals.

Standardized Preparation for Fecal Microbiota Transplantation in Pigs.

The intestine of pigs harbors a mass of microorganisms which are essential for intestinal homeostasis and host health. Intestinal microbial disorders induce enteric inflammation and metabolic dysfunction, thereby causing adverse effects on the growth and health of pigs. In the human medicine, fecal microbiota transplantation (FMT), which engrafts the fecal microbiota from a healthy donor into a patient recipient, has shown efficacy in intestinal microbiota restoration. In addition, it has been used widely in therapy for human gastrointestinal diseases, including Clostridium difficile infection, inflammatory bowel diseases, and irritable bowel syndrome. Given that pigs share many similarities with humans, in terms of anatomy, nutritional physiology, and intestinal microbial compositions, FMT may also be used to restore the normal intestinal microbiota of pigs. However, feasible procedures for performing FMT in pigs remains unclear. Here, we summarize a standardized preparation for FMT in pigs by combining the standard methodology for human FMT with pig production. The key issues include the donor selection, fecal material preparation, fecal material transfer, stool bank establishment, and the safety for porcine FMT. Optimal donors should be selected to ensure the efficacy of porcine FMT and reduce the risks of transmitting infectious diseases to recipients during FMT. Preparing for fresh fecal material is highly recommended. Alternatively, frozen fecal suspension can also be prepared as an optimal choice because it is convenient and has similar efficacy. Oral administration of fecal suspension could be an optimal method for porcine fecal material transfer. Furthermore, the dilution ratio of fecal materials and the frequency of fecal material transfer could be adjusted according to practical situations in the pig industry. To meet the potential large-scale requirement in the pig industry, it is important to establish a stool bank to make porcine FMT readily available. Future studies should also focus on providing more robust safety data on FMT to improve the safety and tolerability of the recipient pigs. This standardized preparation for porcine FMT can facilitate the development of microbial targeted therapies and improve the intestinal health of pigs.

Lactobacillus frumenti Facilitates Intestinal Epithelial Barrier Function Maintenance in Early-Weaned Piglets.

Increased intestinal epithelial barrier function damages caused by early weaning stress have adverse effects on swine health and feed utilization efficiency. Probiotics have emerged as the promising antibiotic alternatives used for intestinal barrier function damage prevention. Our previous data showed that Lactobacillus frumenti was identified as a predominant Lactobacillus in the intestinal microbiota of weaned piglets. However, whether the intestinal epithelial barrier function in piglets was regulated by L. frumenti is still unclear. Here, piglets received a PBS vehicle or PBS suspension (2 ml, 108 CFU/ml) containing the L. frumenti by oral gavage once a day during the period of 6-20 days of age prior to early weaning. Our data demonstrated that oral administration of L. frumenti significantly improved the intestinal mucosal integrity and decreased the serum endotoxin and D-lactic acid levels in early-weaned piglets (26 days of age). The intestinal tight junction proteins (including ZO-1, Occludin, and Claudin-1) were significantly up-regulated by L. frumenti administration. The serum immunoglobulin G (IgG) levels, intestinal secretory immunoglobulin A (sIgA) levels, and interferon-γ (IFN-γ) levels were significantly increased by L. frumenti administration. Furthermore, our data revealed that oral administration of L. frumenti significantly increased the relative abundances of health-promoting microbes (including L. frumenti, Lactobacillus gasseri LA39, Parabacteroides distasonis, and Kazachstania telluris) and decreased the relative abundances of opportunistic pathogens (including Desulfovibrio desulfuricans and Candida humilis). Functional alteration of the intestinal bacterial community by L. frumenti administration was characterized by the significantly increased fatty acids and protein metabolism and decreased diseases-associated metabolic pathways. These findings suggest that L. frumenti facilitates intestinal epithelial barrier function maintenance in early-weaned piglets and may be a promising antibiotic alternative used for intestinal epithelial barrier function damage prevention in mammals.