Junfang Wu

Gut Microbiota Composition Modifies Fecal Metabolic Profiles in Mice

The gut microbiome is known to be extensively involved in human health and disease. In order to reveal the metabolic relationship between host and microbiome, we monitored recovery of the gut microbiota composition and fecal profiles of mice after gentamicin and/or ceftriaxone treatments. This was performed by employing (1)H nuclear magnetic resonance (NMR)-based metabonomics and denaturing gradient gel electrophoresis (DGGE) fingerprint of gut microbiota. The common features of fecal metabolites postantibiotic treatment include decreased levels of short chain fatty acids (SCFAs), amino acids and primary bile acids and increased oligosaccharides, d-pinitol, choline and secondary bile acids (deoxycholic acid). This suggests suppressed bacterial fermentation, protein degradation and enhanced gut microbial modification of bile acids. Barnesiella, Prevotella, and Alistipes levels were shown to decrease as a result of the antibiotic treatment, whereas levels of Bacteroides, Enterococcus and Erysipelotrichaceae incertae sedis, and Mycoplasma increased after gentamicin and ceftriaxone treatment. In addition, there was a strong correlation between fecal profiles and levels of Bacteroides, Barnesiella, Alistipes and Prevotella. The integration of metabonomics and gut microbiota profiling provides important information on the changes of gut microbiota and their impact on fecal profiles during the recovery after antibiotic treatment. The correlation between gut microbiota and fecal metabolites provides important information on the function of bacteria, which in turn could be important in optimizing therapeutic strategies, and developing potential microbiota-based disease preventions and therapeutic interventions.

High-Fat Diet Induces Dynamic Metabolic Alterations in Multiple Biological Matrices of Rats

Obesity is a condition resulting from the interactions of individual biology and environmental factors causing multiple complications. To understand the systems metabolic changes associated with the obesity development and progression, we systematically analyzed the dynamic metabonomic changes induced by a high-fat diet (HFD) in multiple biological matrices of rats using NMR and GC-FID/MS techniques. Clinical chemistry and histopathological data were obtained as complementary information. We found that HFD intakes caused systematic metabolic changes in blood plasma, liver, and urine samples involving multiple metabolic pathways including glycolysis, TCA cycle, and gut microbiota functions together with the metabolisms of fatty acids, amino acids, choline, B-vitamins, purines, and pyrimidines. The HFD-induced metabolic variations were detectable in rat urine a week after HFD intake and showed clear dependence on the intake duration. B-vitamins and gut microbiota played important roles in the obesity development and progression together with changes in TCA cycle intermediates (citrate, α-ketoglutarate, succinate, and fumarate). 83-day HFD intakes caused significant metabolic alterations in rat liver highlighted with the enhancements in lipogenesis, lipid accumulation and lipid oxidation, suppression of glycolysis, up-regulation of gluconeogenesis and glycogenesis together with altered metabolisms of choline, amino acids and nucleotides. HFD intakes reduced the PUFA-to-MUFA ratio in both plasma and liver, indicating the HFD-induced oxidative stress. These findings provided essential biochemistry information about the dynamic metabolic responses to the development and progression of HFD-induced obesity. This study also demonstrated the combined metabonomic analysis of multiple biological matrices as a powerful approach for understanding the molecular basis of pathogenesis and disease progression.

An optimised sample preparation method for NMR-based faecal metabonomic analysis

Faecal metabonomic NMR analysis plays an essential role in investigating the interactions between mammalian metabolism and symbiotic gut microbiota. However, the faecal metabolite extraction method remains to be optimised and standardised to take into consideration signal-to-noise ratios, pH and chemical shift consistency. In the current investigation, we compared extraction consistency of three homogenisation methods including manual ultrasonication, automatic homogenization with tissuelyser and their combination, and systematically optimised faecal metabolite extraction parameters, including the faeces-to-buffer ratio (W(f) : V(b)), extraction repetition times and duration. We found that automatic homogenisation with tissuelyser was the choice of extraction method owning to its good metabolite extraction consistency and high throughput. We also recommend W(f) : V(b) of 1 : 10 (mg microl(-1)) and use of the combined first two extracts as the resultant samples to represent faecal metabolite composition. Such recommendation is based on considerations of maximisation of the spectral signal-to-noise ratio, pH and chemical shift consistency, completeness of metabolite extraction and sample preparation throughput so that the method is suitable for analysing a large number of samples especially in human population studies.

Metabolic alterations in the hamster co-infected with Schistosoma japonicum and Necator americanus

Co-infection with hookworm and schistosomes is a common phenomenon in sub-Saharan Africa, as well as in parts of South America and southeast Asia. As a first step towards understanding the metabolic response of a hookworm-schistosome co-infection in humans, we investigated the metabolic consequences of co-infection in an animal model, using a nuclear magnetic resonance (NMR)-based metabolic profiling technique, combined with multivariate statistical analysis. Urine and serum samples were obtained from hamsters experimentally infected with 250 Necator americanus infective L(3) and 100 Schistosoma japonicum cercariae simultaneously. In the co-infection model, similar worm burdens were observed as reported for single infection models, whereas metabolic profiles of co-infection represented a combination of the altered metabolite profiles induced by single infections with these two parasites. Consistent differences in metabolic profiles between the co-infected and non-infected control hamsters were observed from 4 weeks p.i. onwards. The predominant metabolic alterations in co-infected hamsters consisted of depletion of amino acids, tricarboxylic acid cycle intermediates (e.g. citrate and succinate) and glucose. Moreover, alterations of a series of gut microbial-related metabolites, such as decreased levels of hippurate, 3-hydroxyphenylpropionic acid, 4-hydroxyphenylpropionic acid and trimethylamine-N-oxide, and increased concentrations of 4-cresol glucuronide and phenylacetylglycine were associated with co-infection. Our results provide a first step towards understanding the metabolic response of an animal host to multiple parasitic infections.

Streptozotocin-Induced Dynamic Metabonomic Changes in Rat Biofluids

Diabetes mellitus is a complex polygenic disease caused by gene-environment interactions with multiple complications, and metabonomic analysis is crucial for pathogenesis, early diagnosis, and timely interventions. Here, we comprehensively analyzed the dynamic metabolic changes in rat urine and plasma, which were induced by the well-known diabetogenic chemical streptozotocin (STZ), using (1)H NMR spectroscopy in conjunction with multivariate data analysis. The results showed that a single intraperitoneal injection of STZ with a moderate dosage (55 mg/kg) induced significant urinary metabonomic changes within 24 h. These changes showed time-dependence and heterogeneity among the treated animals with an animal recovered within 11 days. STZ-induced metabonomic alterations were related to suppression of glycolysis and TCA cycle, promotion of gluconeogenesis and oxidation of amino acids, alterations in metabolisms of basic amino acids associated with diabetic complications, and disruption of lipid metabolism and gut microbiota functions. With diffusion-edited NMR spectral data, we further observed the STZ-induced significant elevation of monounsaturated fatty acids and total unsaturated fatty acids together with reductions in PUFA-to-MUFA ratio in the blood plasma. These findings provided details of the time-dependent metabonomic changes in the progressive development of the STZ-induced diabetes mellitus and showed the possibility of detecting the biochemical changes in the early stage of type 1 diabetic genesis.

Metabolic Changes Reveal the Development of Schistosomiasis in Mice

Schistosomiasis is a parasitic zoonosis caused by small trematode worms called schistosomes, amongst which Schistosoma japonicum (S. japonicum) is endemic in Asia. In order to understand the schistosome-induced changes in the host metabolism so as to facilitate early diagnosis of schistosomiasis, we systematically investigated the dynamic metabolic responses of mice biofluids and liver tissues to S. japonicum infection for five weeks using 1H NMR spectroscopy in conjunction with multivariate data analysis. We were able to detect schistosomiasis at the third week post-infection, which was one week earlier than “gold standard” methods. We found that S. japonicum infection caused significant elevation of urinary 3-ureidopropionate, a uracil catabolic product, and disturbance of lipid metabolism, stimulation of glycolysis, depression of tricarboxylic acid cycle and disruption of gut microbiota regulations. We further found that the changes of 3-ureidopropionate and overall metabolic changes in both urinary and plasma samples were closely correlated with the time-course of disease progression. Furthermore, such changes together with liver tissue metabonome were clearly associated with the worm-burdens. These findings provided more insightful understandings of host biological responses to the infection and demonstrated that metabonomic analysis is potentially useful for early detection of schistosomiasis and comprehension of the mechanistic aspects of disease progression.

Advances in metabolic profiling of experimental nematode and trematode infections.

Metabonomics, which is the combination of metabolic profiling of biological samples using spectroscopic methods, together with multivariate data analysis, is a powerful approach for biomarker recovery. Moreover, metabonomics holds promise to enhance our understanding of host-parasite interactions at the metabolic level, and therefore provides a framework for discovery of novel targets for diagnostics, drugs and vaccines. In this review, we summarise progress made to date with metabolic profiling strategies applied to different host-parasite models in the laboratory. First, we emphasise the application of two parasitic worm infections that are particularly relevant for Southeast Asia and the Peoples Republic of China, namely the trematode Schistosoma japonicum causing schistosomiasis, and the nematode Necator americanus causing hookworm disease. Next, we review metabolic profiling studies on the liver fluke Fasciola hepatica in the rat model, the intestinal fluke Echinostoma caproni harboured in mice and characterise the metabolic responses in the hamster to a S. japonicum-N. americanus co-infection. We extract parasite-specific biomarkers and distinguish them from a more general response to an infection at the biochemical level. For example, suppression of tricarboxylic acid cycle metabolites is only noted for a Schistosoma spp. infection, whereas alterations in metabolites derived from the gut microbiota are common for all the parasitic infections investigated thus far. Finally, we explore how the insight gained with experimental infections could be transferred to human populations and conclude with a section on research needs with regard to molecular diagnostics in parasitology.

Gender differences of peripheral plasma and liver metabolic profiling in APP/PS1 transgenic AD mice

Alzheimers disease (AD) is a neurodegenerative disorder characterized by progressive cognitive impairment. Currently, there is less knowledge of the involvement of the peripheral biofluid/organ in AD, compared with the central nervous system. In addition, with reported high morbidity in women in particular, it has become very important to explore whether gender difference in the peripheral metabolome is associated with AD. Here, we investigated metabolic responses of both plasma and liver tissues using an APP/PS1 double mutant transgenic mouse model with NMR spectroscopy, as well as analysis from serum biochemistry and histological staining. Fatty acid composition from plasma and liver extracts was analyzed using GC-FID/MS. We found clear gender differences in AD transgenic mice when compared with their wild-type counterparts. Female AD mice displayed more intensive responses, which were highlighted by higher levels of lipids, 3-hydroxybutyrate and nucleotide-related metabolites, together with lower levels of glucose. These observations indicate that AD induces oxidative stress and impairs cellular energy metabolism in peripheral organs. Disturbances in AD male mice were milder with depletion of monounsaturated fatty acids. We also observed a higher activity of delta-6-desaturate and suppressed activity of delta-5-desaturate in female mice, whereas inhibited stearoyl-CoA-desaturase in male mice suggested that AD induced by the double mutant genes results in different fatty acids catabolism depending on gender. Our results provide metabolic clues into the peripheral biofluid/organs involved in AD, and we propose that a gender-specific scheme for AD treatment in men and women may be required.

1H-NMR Spectroscopy Revealed Mycobacterium tuberculosis Caused Abnormal Serum Metabolic Profile of Cattle

To re-evaluate virulence of Mycobacterium tuberculosis (M. tb) in cattle, we experimentally infected calves with M. tb andMycobacterium bovisvia intratracheal injection at a dose of 2.0×107 CFU and observed the animals for 33 weeks. The intradermal tuberculin test and IFN-γin vitro release assay showed that both M. tb and M. bovis induced similar responses. Immunohistochemical staining of pulmonary lymph nodes indicated that the antigen MPB83 of both M. tb and M. bovis were similarly distributed in the tissue samples. Histological examinations showed all of the infected groups exhibited neutrophil infiltration to similar extents. Although the infected cattle did not develop granulomatous inflammation, the metabolic profiles changed significantly, which were characterized by a change in energy production pathways and increased concentrations of N-acetyl glycoproteins. Glycolysis was induced in the infected cattle by decreased glucose and increased lactate content, and enhanced fatty acid β-oxidation was induced by decreased TG content, and decreased gluconeogenesis indicated by the decreased concentration of glucogenic and ketogenic amino acids promoted utilization of substances other than glucose as energy sources. In addition, an increase in acute phase reactive serum glycoproteins, together with neutrophil infiltration and increased of IL-1β production indicated an early inflammatory response before granuloma formation. In conclusion, this study indicated that both M. tb and M.bovis were virulent to cattle. Therefore, it is likely that cattle with M. tb infections would be critical to tuberculosis transmission from cattle to humans. Nuclear magnetic resonance was demonstrated to be an efficient method to systematically evaluate M. tb and M. bovi sinfection in cattle.

Metabolomics Insights into the Modulatory Effects of Long-Term Low Calorie Intake in Mice

There is increasing evidence that calorie restriction without malnutrition can extend longevity and delay the onset of age-associated disorders. Identifying the biochemical perturbations associated with different dietary habits would provide valuable insights into associations between metabolism and longevity. To reveal the effects of long-term dietary interventions on metabolic perturbations, we investigated serum and urinary metabolic changes induced by interactive high/low fat diet in combination with/without reduced caloric intake over a life span in mice using NMR-based metabonomics. We found that the high calorie dietary regime disturbed lipid metabolism, suppressed glycolysis and TCA cycles, stimulated oxidative stress, promoted nucleotide metabolism and gluconeogenesis, and perturbed gut microbiota-host interactions. Such changes could be modified by long-term low calorie intake. Most importantly, we found that the calorie intake index exerts a dominant effect on metabolic perturbations irrespective of dietary regime. Our investigation provides a holistic view of the metabolic impact of long-term dietary interventions, which are important for detecting physiological changes and dietary effects on mammalian metabolism.