Hippurate, the glycine conjugate of benzoic acid, is a normal constituent of the endogenous urinary metabolite profile and has long been associated with the microbial degradation of certain dietary components, hepatic function and toluene exposure, and is also commonly used as a measure of renal clearance. Here we discuss the potential relevance of hippurate excretion with regard to normal endogenous metabolism and trends in excretion relating to gender, age, and the intestinal microbiota. Additionally, the significance of hippurate excretion with respect to disease states including obesity, diabetes, gastrointestinal diseases, impaired renal function, psychological disorders and autism, as well as toxicity and parasitic infection, are considered.

The altered Schaedler flora (ASF) is a model microbial community with both in vivo and in vitro relevance. Here we provide the first characterization of the ASF community in vitro, independent of a murine host. We compared the functional genetic content of the ASF to wild murine metagenomes and found that the ASF functionally represents wild microbiomes better than random consortia of similar taxonomic composition. We developed a chemically defined medium that supported growth of seven of the eight ASF members. To elucidate the metabolic capabilities of these ASF species—including potential for interactions such as cross-feeding—we performed a spent media screen and analyzed the results through dynamic growth measurements and non-targeted metabolic profiling. We found that cross-feeding is relatively rare (32 of 3570 possible cases), but is enriched between Clostridium ASF356 and Parabacteroides ASF519. We identified many cases of emergent metabolism (856 of 3570 possible cases). These data will inform efforts to understand ASF dynamics and spatial distribution in vivo, to design pre- and probiotics that modulate relative abundances of ASF members, and will be essential for validating computational models of ASF metabolism. Well-characterized, experimentally tractable microbial communities enable research that can translate into more effective microbiome-targeted therapies to improve human health.

Animal models are invaluable tools which allow us to investigate the microbiome-host dialogue. However, experimental design introduces biases in the data that we collect, also potentially leading to biased conclusions. With obesity at pandemic levels animal models of this disease have been developed; we investigated the role of experimental design on one such rodent model. We used 454 pyrosequencing to profile the faecal bacteria of obese (n = 6) and lean (homozygous n = 6; heterozygous n = 6) Zucker rats over a 10 week period, maintained in mixed-genotype cages, to further understand the relationships between the composition of the intestinal bacteria and age, obesity progression, genetic background and cage environment. Phylogenetic and taxon-based univariate and multivariate analyses (non-metric multidimensional scaling, principal component analysis) showed that age was the most significant source of variation in the composition of the faecal microbiota. Second to this, cage environment was found to clearly impact the composition of the faecal microbiota, with samples from animals from within the same cage showing high community structure concordance, but large differences seen between cages. Importantly, the genetically induced obese phenotype was not found to impact the faecal bacterial profiles. These findings demonstrate that the age and local environmental cage variables were driving the composition of the faecal bacteria and were more deterministically important than the host genotype. These findings have major implications for understanding the significance of functional metagenomic data in experimental studies and beg the question; what is being measured in animal experiments in which different strains are housed separately, nature or nurture?

Facilitative urea transporters in the mammalian kidney play a vital role in the urinary concentrating mechanism. The urea transporters located in the renal inner medullary collecting duct, namely UT-A1 and UT-A3, are acutely regulated by the antidiuretic hormone vasopressin. In this study, we investigated the vasopressin regulation of the basolateral urea transporter UT-A3 using an MDCK-mUT-A3 cell line. Within 10 min, vasopressin stimulates urea flux through UT-A3 transporters already present at the plasma membrane, via a PKA-dependent process. Within 1 h, vasopressin significantly increases UT-A3 localization at the basolateral membrane, causing a further increase in urea transport. While the basic trafficking of UT-A3 to basolateral membranes involves both protein kinase C and calmodulin, its regulation by vasopressin specifically occurs through a casein kinase II-dependent pathway. In conclusion, this study details the effects of vasopressin on UT-A3 urea transporter function and hence its role in regulating urea permeability within the renal inner medullary collecting duct. arginine vasopressin; urea transport; membrane localization; casein kinase II IN MAMMALS, THE PASSIVE MOVEMENT of urea across cell membranes is facilitated by phloretin-sensitive urea transporters derived from two distinct genes, UT-A (Slc14a2) and UT-B (Slc14a1) (20). In the kidney, there are four major isoforms of these facilitative urea transporters, three are derived from alternative splicing of the UT-A gene (namely UT-A1, UT-A2, and UT-A3), plus one UT-B isoform (UT-B1) (22). Renal facilitative urea transporters are vital to the urinary concentrating mechanism and mouse knockout models have shown that UT-A1 and UT-A3 (8), UT-A2 (27), and UT-B1 (1, 29) all play a significant role in producing concentrated urine.UT-A1 and UT-A3 are both located in the inner medullary collecting duct (IMCD) (7,21,24). Indeed, it has now been shown that the UT-A urea transporter promoter, UT-A alpha, is responsible for this specific targeting to principal cells of the renal IMCD (10). At a subcellular level, UT-A1 is located to the apical membrane of IMCD cells (18). In contrast, the UT-A3 isoform, originally cloned from rat kidney (15), has been localized to the basolateral membrane in both mouse (24) and rat (17). In combination, UT-A1 and UT-A3 are responsible for urea reabsorption from the IMCD lumen to the inner medullary interstitium, and the absence of these transporters causes a urea-dependent osmotic diuresis (9).In perfused isolated IMCD, vasopressin stimulates phloretin-sensitive trans-epithelial urea transport (23,28). Recent studies of rat UT-A1 (11) and mouse UT-A3 (25) expressed in Madin-Darby canine kidney (MDCK) type I cell lines showed that both isoforms can be acutely regulated by arginine vasopressin (AVP), via a cAMP-dependent pathway. Interestingly, the protein kinase A (PKA) inhibitor H89 reduced the vasopressin activation of both UT-A1 (12) and UT-A3 (25). Vasopressin is known to rapidly increase phosphorylation of UT-A1 via PKA...

No abstract

Hyperpolarized [1‐13C] pyruvate can be used to examine the metabolic state of cancer cells, highlighting a key metabolic characteristic of cancer: the upregulated metabolic flux to lactate, even in the presence of oxygen (Warburg effect). Thus, the rate constant of 13C exchange of pyruvate to lactate, kPL, can serve as a metabolic biomarker of cancer presence, aggressiveness and therapy response. Established in vitro hyperpolarized experiments dissolve the probe for each cell sample independently, an inefficient process that consumes excessive time and resources. Expanding on our previous development of a microcoil with greatly increased detection sensitivity (103‐fold) compared with traditional in vitro methods, we present a novel microcoil equipped with a 10‐μL vertical reservoir and an experimental protocol utilizing deuterated dissolution buffer to measure metabolic flux in multiple mass‐limited cell suspension samples using a single dissolution. This method increases efficiency and potentially reduces the methodological variability associated with hyperpolarized experiments. This technique was used to measure pyruvate‐to‐lactate flux in melanoma cells to assess BRAF‐inhibition treatment response. There was a significant reduction of kPL in BRAFV600E cells following 24 and 48 hours of treatment with 2 μM vemurafenib (P ≤ .05). This agrees with significant changes observed in the pool sizes of extracellular lactate (P ≤ .05) and glucose (P ≤ .001) following 6 and 48 hours of treatment, respectively, and a significant reduction in cell proliferation following 72 hours of treatment (P ≤ .01). BRAF inhibition had no significant effect on the metabolic flux of BRAFWT cells. These data demonstrate a 6‐8–fold increase in efficiency for the measurement of kPL in cell suspension samples compared with traditional hyperpolarized in vitro methods.

ABSTRACT:The Zucker (fa/fa) rat is a valuable and extensively utilized model for obesity research. However, the metabolic networks underlying the systemic response in the obese Zucker rats remain to be elucidated. This information is important to further our understanding of the circulation of the microbial or host-microbial metabolites and their impact on host metabolism.1 H Nuclear Magnetic Resonance spectroscopy-based metabolic profiling was used to probe global metabolic differences in portal vein and peripheral blood plasma, urine and fecal water between obese (fa/fa, n=12) and lean (fa/+, n=12) Zucker rats. Urinary concentrations of host-microbial co-metabolites were found to be significantly higher in lean Zucker rats. Higher concentrations of fecal lactate, short chain fatty acids (SCFAs), 3-hydroxyphenyl propionic acid and glycerol, and lower levels of valine and glycine were observed in obese rats compared with lean animals. Regardless of phenotype, concentrations of SCFAs, tricarboxylic acid cycle intermediates, and choline metabolites were higher in portal vein blood compared to peripheral blood. However, higher levels of succinate, phenylalanine and tyrosine were observed in portal vein blood compared with peripheral blood from lean rats but not in obese rats. Our findings indicate that the absorption of propionate and acetate, choline and TMA are independent of the Zucker rat phenotypes. However, urinary host-microbial co-metabolites were highly associated with phenotypes, suggesting distinct gut microbial metabolic activities in lean and obese Zucker rats. This work advances our understanding of metabolic processes associated with obesity, particularly the metabolic functionality of the gut microbiota in the context of obesity.

Obesity and its co-morbidities are increasing worldwide imposing a heavy socioeconomic burden. The effects of obesity on the metabolic profiles of tissues (liver, kidney, pancreas), urine and the systemic circulation were investigated in the Zucker rat model using 1 H NMR spectroscopy coupled to multivariate statistical analysis. The metabolic profiles of the obese (fa/fa) animals were clearly differentiated from the two phenotypically lean phenotypes, ((+/+) and (fa/+)) within