Yeon-Ran Kim

Alteration of gut microbiota by vancomycin and bacitracin improves insulin resistance via glucagon-like peptide 1 in diet-induced obesity

Firmicutes and Bacteroidetes, 2 major phyla of gut microbiota, are involved in lipid and bile acid metabolism to maintain systemic energy homeostasis in host. Recently, accumulating evidence has suggested that dietary changes promptly induce the alteration of abundance of both Firmicutes and Bacteroidetes in obesity and its related metabolic diseases. Nevertheless, the metabolic roles of Firmicutes and Bacteroidetes on such disease states remain unclear. The aim of this study was to determine the effects of antibiotic‐induced depletion of Firmicutes and Bacteroidetes on dysregulation of energy homeostasis in obesity. Treatment of C57BL/6J mice with the antibiotics (vancomycin [V] and bacitracin [B]), in the drinking water, before diet‐induced obesity (DIO) greatly decreased both Firmicutes and Bacteroidetes in the gut as revealed by pyrosequencing of the microbial 16S rRNA gene. Concomitantly, systemic glucose intolerance, hyperinsulinemia, and insulin resistance in DIO were ameliorated via augmentation of GLP‐1 secretion (active form; 2.03‐fold, total form; 5.09‐fold) independently of obesity as compared with untreated DIO controls. Furthermore, there were increases in metabolically beneficial metabolites derived from the gut. Together, our data suggest that Firmicutes and Bacteroidetes potentially mediate insulin resistance through modulation of GLP‐1 secretion in obesity.—Hwang, I., Park, Y. J., Kim, Y. ‐R., Kim, Y. N., Ka, S., Lee, H. Y., Seong, J. K., Seok, Y.‐J., Kim, J. B. Alteration of gut microbiota by vancomycin and bacitracin improves insulin resistance via glucagon‐like peptide 1 in diet‐induced obesity. FASEB J. 29, 2397‐2411 (2015). www.fasebj.org

Disruption of γ-glutamylcysteine synthetase results in absolute glutathione auxotrophy and apoptosis in Candida albicans

Glutathione is the most abundant non‐protein thiol and a major source of reducing equivalents in eukaryotes. We examined the role of glutathione in Candida albicans by the disruption of γ‐glutamylcysteine synthetase (GCS1), an essential enzyme in glutathione biosynthesis. The gcs1/gcs1 null mutants exhibited glutathione auxotrophy, which could be rescued by supplementing with reduced and oxidized glutathione and γ‐glutamylcysteine. When the mutants were depleted of glutathione, they showed typical markers of apoptosis. These results suggest that glutathione itself is an essential metabolite and C. albicans lacking GCS1 undergoes apoptosis.

Reciprocal regulation of the autophosphorylation of enzyme INtr by glutamine and α-ketoglutarate in Escherichia coli

In addition to the phosphoenolpyruvate:sugar phosphotransferase system (sugar PTS), most proteobacteria possess a paralogous system (nitrogen phosphotransferase system, PTSNtr). The first proteins in both pathways are enzymes (enzyme Isugar and enzyme INtr) that can be autophosphorylated by phosphoenolpyruvate. The most striking difference between enzyme Isugar and enzyme INtr is the presence of a GAF domain at the N‐terminus of enzyme INtr. Since the PTSNtr was identified in 1995, it has been implicated in a variety of cellular processes in many proteobacteria and many of these regulations have been shown to be dependent on the phosphorylation state of PTSNtr components. However, there has been little evidence that any component of this so‐called PTSNtr is directly involved in nitrogen metabolism. Moreover, a signal regulating the phosphorylation state of the PTSNtr had not been uncovered. Here, we demonstrate that glutamine and α‐ketoglutarate, the canonical signals of nitrogen availability, reciprocally regulate the phosphorylation state of the PTSNtr by direct effects on enzyme INtr autophosphorylation and the GAF signal transduction domain is necessary for the regulation of enzyme INtr activity by the two signal molecules. Taken together, our results suggest that the PTSNtr senses nitrogen availability.

Mitochondrial NADH-cytochrome b5 reductase plays a crucial role in the reduction of d-erythroascorbyl free radical in Saccharomyces cerevisiae

The relevance of NADH-cytochrome b(5) reductase to the NADH-dependent reduction of D-erythroascorbyl free radical was investigated in Saccharomyces cerevisiae. MCR1, which is known to encode NADH-cytochrome b(5) reductase in S. cerevisiae, was disrupted by the insertion of URA3 gene into the gene of MCR1. In the mcr1 disruptant cells, the activity of NADH-D-erythroascorbyl free radical reductase almost disappeared and the intracellular level of D-erythroascorbic acid was about 11% of that of the congenic wild-type strain. In the transformant cells carrying MCR1 in multicopy plasmid, the intracellular level of D-erythroascorbic acid and the activity of NADH-D-erythroascorbyl free radical reductase increased up to 1.7-fold and 2.1-fold, respectively. Therefore, it indicated that the MCR1 product, mitochondrial NADH-cytochrome b(5) reductase, plays a key role in the NADH-dependent reduction of D-erythroascorbyl free radical in S. cerevisiae. On the other hand, the mcr1 disruptant cells were hypersensitive to hydrogen peroxide and menadione, and overexpression of MCR1 made the cells more resistant against oxidative stress. These results suggested that the mitochondrial NADH-cytochrome b(5) reductase functions as NADH-D-erythroascorbyl free radical reductase and plays an important role in the response to oxidative damage in S. cerevisiae.

Analysis of Gastric Microbiota by Pyrosequencing: Minor Role of Bacteria Other Than Helicobacter pylori in the Gastric Carcinogenesis.

Little is known about the role of gastric microbiota except for Helicobacter pylori (HP) in human health and disease. We compared the differences of human gastric microbiota according to gastric cancer or control and HP infection status and assessed the role of bacteria other than HP.

RppH-dependent pyrophosphohydrolysis of mRNAs is regulated by direct interaction with DapF in Escherichia coli.

Similar to decapping of eukaryotic mRNAs, the RppH-catalyzed conversion of 5′-terminal triphosphate to monophosphate has recently been identified as the rate-limiting step for the degradation of a subset of mRNAs in Escherichia coli. However, the regulation of RppH pyrophosphohydrolase activity is not well understood. Because the overexpression of RppH alone does not affect the decay rate of most target mRNAs, the existence of a mechanism regulating its activity has been suggested. In this study, we identified DapF, a diaminopimelate (DAP) epimerase catalyzing the stereoinversion of L,L-DAP to meso-DAP, as a regulator of RppH. DapF showed a high affinity interaction with RppH and increased its RNA pyrophosphohydrolase activity. The simultaneous overexpression of both DapF and RppH increased the decay rates of RppH target RNAs by about a factor of two. Together, our data suggest that the cellular level of DapF is a critical factor regulating the RppH-catalyzed pyrophosphate removal and the subsequent degradation of target mRNAs.

An Appropriate Cutoff Value for Determining the Colonization of Helicobacter pylori by the Pyrosequencing Method: Comparison with Conventional Methods

Sequencing of 16S ribosomal RNA (rRNA) gene has improved the characterization of microbial communities. It enabled the detection of low abundance gastric Helicobacter pylori sequences even in subjects that were found to be H. pylori negative with conventional methods. The objective of this study was to obtain a cutoff value for H. pylori colonization in gastric mucosa samples by pyrosequencing method.

Comparison of Gastric Microbiota Between Gastric Juice and Mucosa by Next Generation Sequencing Method

Background: Not much is known about the role of gastric microbiota except for Helicobacter pylori in human health and disease. In this study, we aimed to detect human gastric microbiota in both gastric mucosa and gastric juice by barcoded 454-pyrosequencing of the 16S rRNA gene and to compare the results from mucosa and juice. Methods: Gastric biopsies and stomach juices were collected from 4 subjects who underwent standard endoscopy at Seoul National University Bundang Hospital. Gastric microbiota of antral mucosa, corpus mucosa samples, and gastric fluids were analyzed by barcoded 454-pyrosequencing of the 16S rRNA gene. The analysis focused on bacteria, such as H. pylori and nitrosating or nitrate-reducing bacteria. Results: Gastric fluid samples showed higher diversity compared to that of gastric mucosa samples. The mean of operational taxonomic units was higher in gastric fluid than in gastric mucosa. The samples of gastric fluid and gastric mucosa showed different composition of phyla. The composition of H. pylori and Proteobacteria was higher in mucosa samples compared to gastric fluid samples (H. pylori, 66.5% vs. 3.3%, P = 0.033; Proteobacteria, 75.4% vs. 26.3%, P = 0.041), while Actinobacteria, Bacteroidetes, and Firmicutes were proportioned relatively less in mucosa samples than gastric fluid. However there was no significant difference. (Actinobacteria, 3.5% vs. 20.2%, P = 0.312; Bacteroidetes, 6.0% vs. 14.8%, P = 0.329; Firmicutes, 12.8% vs. 33.4%, P = 0.246). Conclusions: Even though these samples were small, gastric mucosa could be more effective than gastric fluid in the detection of meaningful gastric microbiota by pyrosequencing.

Immunomodulatory Activity of the Water Extract from Medicinal Mushroom Inonotus obliquus.

The immunomodulatory effect of aqueous extract of Inonotus obliquus, called as Chaga, was tested on bone marrow cells from chemically immunosuppressed mice. The Chaga water extract was daily administered for 24 days to mice that had been treated with cyclophosphamide (400 mg/kg body weight), immunosuppressive alkylating agent. The number of colony-forming unit (CFU)-granuloeytes/maerophages (GM) and erythroid burst-forming unit (BFU-E), increased almost to the levels seen in non-treated control as early as 8 days after treatment. Oral administration of the extract highly increased serum levels of IL-6. Also, the level of TNF-α was elevated by the chemical treatment in control mice, whereas was maintained at the background level in the extract-treated mice, indicating that the extract might effectively suppress TNF-α related pathologic conditions. These results strongly suggest the great potential of the aqueous extract from Inonotus obliquus as immune enhancer during chemotherapy.

Impact of Long-Term Proton Pump Inhibitor Therapy on Gut Microbiota in F344 Rats: Pilot Study.

Background/Aims To evaluate changes in gut microbiota composition following long-term proton pump inhibitor (PPI) treatment. Methods Twenty-four-week-old F344 rats were fed diets with (n=6) or without (n=5) lansoprazole for 50 weeks. Profiles of luminal microbiota in the terminal ileum were then analyzed. Pyrosequencing of the 16S rRNA gene was performed using an FLX genome sequencer (454 Life Sciences/Roche). Results Rats treated with lansoprazole showed significantly reduced body weights compared to controls (lansoprazole-treated rats and controls, 322.3±15.3 g vs 403.2±5.2 g, respectively, p<0.001). However, stool frequencies and consistencies did not differ between the two groups. The composition of the gut microbiota in lansoprazole-treated rats was quite different from that of the controls. In the controls, the microbiota profiles obtained from the terminal ileum showed a predominance of Proteobacteria (93.9%) due to the abundance of Escherichia and Pasteurella genera. Conversely, lansoprazole-treated rats showed an elevated population of Firmicutes (66.9%), which was attributed to an increased ratio of Clostridium g4 to Lactobacillus genera. Conclusions This preliminary study suggests that long-term administration of PPI may cause weight loss and changes to the microbiota in the terminal ileum.