The gut microbiota regulates hypothalamic inflammation and leptin sensitivity in Western diet-fed mice via a GLP-1R-dependent mechanism.

Mice lacking a microbiota are protected from diet-induced obesity. Previous studies have shown that feeding a Western diet causes hypothalamic inflammation, which in turn can lead to leptin resistance and weight gain. Here, we show that wild-type (WT) mice with depleted gut microbiota, i.e., germ-free (GF) and antibiotic-treated mice, have elevated levels of glucagon-like peptide-1 (GLP-1), are protected against diet-induced hypothalamic inflammation, and have enhanced leptin sensitivity when fed a Western diet. Using GLP-1 receptor (GLP-1R)-deficient mice and pharmacological inhibition of the GLP-1R in WT mice, we demonstrate that intact GLP-1R signaling is required for preventing hypothalamic inflammation and enhancing leptin sensitivity. Furthermore, we show that astrocytes express the GLP-1R, and deletion of the receptor in glial fibrillary acidic protein (GFAP)-expressing cells diminished the antibiotic-induced protection against diet-induced hypothalamic inflammation. Collectively, our results suggest that depletion of the gut microbiota attenuates diet-induced hypothalamic inflammation and enhances leptin sensitivity via GLP-1R-dependent mechanisms.

Microbially produced glucagon-like peptide 1 improves glucose tolerance in mice.

OBJECTIVE: The enteroendocrine hormone glucagon-like peptide 1 (GLP-1) is an attractive anti-diabetic therapy. Here, we generated a recombinant Lactococcus lactis strain genetically modified to produce GLP-1 and investigated its ability to improve glucose tolerance in mice on chow or high-fat diet (HFD). METHODS: We transformed L. lactis FI5876 with either empty vector (pUK200) or murine GLP-1 expression vector to generate LL-UK200 and LL-GLP1, respectively, and determined their potential to induce insulin secretion by incubating primary islets from wild-type (WT) and GLP-1 receptor knockout (GLP1R-KO) mice with culture supernatant of these strains. In addition, we administered these strains to mice on chow or HFD. At the end of the study period, we measured plasma GLP-1 levels, performed intraperitoneal glucose tolerance and insulin tolerance tests, and determined hepatic expression of the gluconeogenic genes G6pc and Pepck. RESULTS: Insulin release from primary islets of WT but not GLP1R-KO mice was higher following incubation with culture supernatant from LL-GLP1 compared with LL-UK200. In mice on chow, supplementation with LL-GLP1 versus LL-UK200 promoted increased vena porta levels of GLP-1 in both WT and GLP1R-KO mice; however, LL-GLP1 promoted improved glucose tolerance in WT but not in GLP1R-KO mice, indicating a requirement for the GLP-1 receptor. In mice on HFD and thus with impaired glucose tolerance, supplementation with LL-GLP1 versus LL-UK200 promoted a pronounced improvement in glucose tolerance together with increased insulin levels. Supplementation with LL-GLP1 versus LL-UK200 did not affect insulin tolerance but resulted in reduced expression of G6pc in both chow and HFD-fed mice. CONCLUSIONS: The L. lactis strain genetically modified to produce GLP-1 is capable of stimulating insulin secretion from islets and improving glucose tolerance in mice.

Insulin-like peptide 5 is a microbially regulated peptide that promotes hepatic glucose production.

OBJECTIVE: Insulin-like peptide 5 (INSL5) is a recently identified gut hormone that is produced predominantly by L-cells in the colon, but its function is unclear. We have previously shown that colonic expression of the gene for the L-cell hormone GLP-1 is high in mice that lack a microbiota and thus have energy-deprived colonocytes. Our aim was to investigate if energy deficiency also affected colonic Insl5 expression and to identify a potential role of INSL5. METHODS: We analyzed colonic Insl5 expression in germ-free (GF), conventionally raised (CONV-R), conventionalized (CONV-D) and antibiotic-treated mice, and also assessed the effect of dietary changes on colonic Insl5 expression. In addition, we characterized the metabolic phenotype of Insl5-/- mice. RESULTS: We showed that colonic Insl5 expression was higher in GF and antibiotic-treated mice than in CONV-R mice, whereas Insl5 expression in the brain was higher in CONV-R versus GF mice. We also observed that colonic Insl5 expression was suppressed by increasing the energy supply in GF mice by colonization or high-fat feeding. We did not observe any differences in food intake, gut transit or oral glucose tolerance between Insl5-/- and wild-type mice. However, we showed impaired intraperitoneal glucose tolerance in Insl5-/- mice. We also observed improved insulin tolerance and reduced hepatic glucose production in Insl5-/- mice. CONCLUSIONS: We have shown that colonic Insl5 expression is regulated by the gut microbiota and energy availability. We propose that INSL5 is a hormone that could play a role in promoting hepatic glucose production during periods of energy deprivation.

Dietary Fiber-Induced Improvement in Glucose Metabolism Is Associated with Increased Abundance of Prevotella.

The gut microbiota plays an important role in human health by interacting with host diet, but there is substantial inter-individual variation in the response to diet. Here we compared the gut microbiota composition of healthy subjects who exhibited improved glucose metabolism following 3-day consumption of barley kernel-based bread (BKB) with those who responded least to this dietary intervention. The Prevotella/Bacteroides ratio was higher in responders than non-responders after BKB. Metagenomic analysis showed that the gut microbiota of responders was enriched in Prevotella copri and had increased potential to ferment complex polysaccharides after BKB. Finally, germ-free mice transplanted with microbiota from responder human donors exhibited improved glucose metabolism and increased abundance of Prevotella and liver glycogen content compared with germ-free mice that received non-responder microbiota. Our findings indicate that Prevotella plays a role in the BKB-induced improvement in glucose metabolism observed in certain individuals, potentially by promoting increased glycogen storage.

Dynamics and Stabilization of the Human Gut Microbiome during the First Year of Life.

The gut microbiota is central to human health, but its establishment in early life has not been quantitatively and functionally examined. Applying metagenomic analysis on fecal samples from a large cohort of Swedish infants and their mothers, we characterized the gut microbiome during the first year of life and assessed the impact of mode of delivery and feeding on its establishment. In contrast to vaginally delivered infants, the gut microbiota of infants delivered by C-section showed significantly less resemblance to their mothers. Nutrition had a major impact on early microbiota composition and function, with cessation of breast-feeding, rather than introduction of solid food, being required for maturation into an adult-like microbiota. Microbiota composition and ecological network had distinctive features at each sampled stage, in accordance with functional maturation of the microbiome. Our findings establish a framework for understanding the interplay between the gut microbiome and the human body in early life.

Evaluation of meisoindigo, an indirubin derivative: in vitro antileukemic activity and in vivo pharmacokinetics.

Meisoindigo has been a routine therapeutic agent in the clinical treatment of chronic myelogenous leukemia (CML) in China since the 1980s. In the present study, the in vitro antileukemic activity of meisoindigo was investigated in acute promyelocytic leukemia (APL) cells, acute myeloid leukemia (AML) cells, and myelomonocytic leukemia cells (NB4, NB4.007/6, HL-60 and U937) comprising both retinoic acid-sensitive and retinoic acid-resistant cells. We found that meisoindigo effectively inhibited the growth and/or proliferation of these four cell types at µM levels. The effects of meisoindigo in these cells are related to its proliferation inhibition and apoptosis induction, and are independent of cell cycle arrest, indicating that meisoindigo could be possible in the treatment of APL, AML and retinoic acid resistant APL. The in vivo pharmacokinetics of meisoindigo and its major circulatory metabolites in rat plasma were then investigated by a newly developed and validated liquid chromatography-tandem mass spectrometry (LC-MS/MS) method. The profiles of plasma concentration versus time were plotted and the relevant pharmacokinetic parameters were calculated for meisoindigo and its reductive metabolites. The plasma concentrations of meisoindigo after oral administration were much lower than the in vitro IC50s determined in the leukemic cells. The contradicting poor pharmacokinetic characteristics and the established clinical efficacy of meisoindigo could indicate the presence of active metabolites in vivo.

Analysis of gut microbial regulation of host gene expression along the length of the gut and regulation of gut microbial ecology through MyD88.

BACKGROUND: The gut microbiota has profound effects on host physiology but local host-microbial interactions in the gut are only poorly characterised and are likely to vary from the sparsely colonised duodenum to the densely colonised colon. Microorganisms are recognised by pattern recognition receptors such as Toll-like receptors, which signal through the adaptor molecule MyD88. METHODS: To identify host responses induced by gut microbiota along the length of the gut and whether these required MyD88, transcriptional profiles of duodenum, jejunum, ileum and colon were compared from germ-free and conventionally raised wild-type and Myd88-/- mice. The gut microbial ecology was assessed by 454-based pyrosequencing and viruses were analysed by PCR. RESULTS: The gut microbiota modulated the expression of a large set of genes in the small intestine and fewer genes in the colon but surprisingly few microbiota-regulated genes required MyD88 signalling. However, MyD88 was essential for microbiota-induced colonic expression of the antimicrobial genes Reg3ß and Reg3γ in the epithelium, and Myd88 deficiency was associated with both a shift in bacterial diversity and a greater proportion of segmented filamentous bacteria in the small intestine. In addition, conventionally raised Myd88-/- mice had increased expression of antiviral genes in the colon, which correlated with norovirus infection in the colonic epithelium. CONCLUSION: This study provides a detailed description of tissue-specific host transcriptional responses to the normal gut microbiota along the length of the gut and demonstrates that the absence of MyD88 alters gut microbial ecology.

Identification of circulatory and excretory metabolites of meisoindigo in rat plasma, urine and feces by high-performance liquid chromatography coupled with positive electrospray ionization tandem mass spectrometry.

Meisoindigo has been a routine therapeutic agent in the clinical treatment of chronic myelogenous leukemia in China since the 1980s. However, information relevant to in vivo metabolism of meisoindigo is absent so far. In this study, in vivo circulatory metabolites of meisoindigo in rat plasma, as well as excretory metabolites in rat urine and feces, were identified by liquid chromatography/tandem mass spectrometry (LC/MS/MS). Integration of multiple reaction monitoring with conventional metabolic profiling methodology was adopted to enable a more sensitive detection of in vivo metabolites. By comparing with the MS/MS spectra and retention times of the in vitro reduced metabolites, the major metabolites in rat plasma were proposed to form from 3,3' double bond reduction, whereas the minor metabolites were formed from reduction followed by N-demethylation, and reduction followed by phenyl mono-oxidation. The major metabolites in the rat urine were proposed to form from reduction followed by phenyl mono-oxidation, and its glucuronide conjugation and sulfate conjugation, whereas the minor metabolites were formed from 3,3' double bond reduction, N-demethylation, reduction followed by N-demethylation, phenyl di-oxidation, phenyl mono-oxidation and its glucuronide conjugation and sulfate conjugation. The major metabolites in the rat feces were proposed to form from reduction followed by phenyl mono-oxidation, whereas the minor metabolites were formed from reduction followed by N-demethylation, and reduction followed by phenyl di-oxidation. The phase I metabolic pathways showed a significant in vitro-in vivo correlation in rat.