Improvement of start-up and nitrogen removal of the anammox process in reactors inoculated with conventional activated sludge using biofilm carrier materials

<p>The start-up of the anaerobic ammonium oxidation (anammox) process in three up-flow column reactors seeded with common mixed activated sludge and added with three materials, sponge (R1), sponge + volcanic rock (R2) and sponge + charcoal (R3), as carriers for biofilm formation were comparatively investigated in this study. The supplement of volcanic rock and charcoal could significantly shorten the start-up time of the anammox process, which primarily occurred in the activity-enhanced phase, with ammonium and nitrite removal efficiencies stabilized above 92.5% and 93.4% after an operation period of 145, 105 and 121 d for R1, R2 and R3, respectively. After the successful anammox start-up, R2 performed significantly better in TN removal (<i>p </i>< .05), achieving an average rate of 91.0% and 191.5 g N m⁻³ d⁻¹ compared to R1 of 88.4% and 172.1 g N m⁻³ d⁻¹, and R3 of 89.9% and 180.1 g N m⁻³ d⁻¹ in the steady running phase. The ratios of consumed and generated /consumed after anammox start-up were lower than the theoretical values, probably suggesting the simultaneous existences of anammox, denitrification as well as nitrification processes in the reactors. A reddish brown biofilm was wrapped on the carriers and morphological detection of biofilm displayed the presentations of thick and compact floc aggregates and some filamentous bacteria on the sponge, and spherical-, ovoid- and shortrod-shaped microorganisms on the volcanic rock and charcoal. Using porous material as carrier for biofilm development is an effective strategy for practical application of the anammox reactor.</p

A Potent Class of GPR40 Full Agonists Engages the EnteroInsular Axis to Promote Glucose Control in Rodents

<div><p>Type 2 diabetes is characterized by impaired glucose homeostasis due to defects in insulin secretion, insulin resistance and the incretin response. GPR40 (FFAR1 or FFA1) is a G-protein-coupled receptor (GPCR), primarily expressed in insulin-producing pancreatic β-cells and incretin-producing enteroendocrine cells of the small intestine. Several GPR40 agonists, including AMG 837 and TAK-875, have been disclosed, but no GPR40 synthetic agonists have been reported that engage both the insulinogenic and incretinogenic axes. In this report we provide a molecular explanation and describe the discovery of a unique and potent class of GPR40 full agonists that engages the enteroinsular axis to promote dramatic improvement in glucose control in rodents. GPR40 full agonists AM-1638 and AM-6226 stimulate GLP-1 and GIP secretion from intestinal enteroendocrine cells and increase GSIS from pancreatic islets, leading to enhanced glucose control in the high fat fed, streptozotocin treated and NONcNZO10/LtJ mouse models of type 2 diabetes. The improvement in hyperglycemia by AM-1638 was reduced in the presence of the GLP-1 receptor antagonist Ex(9–39)NH₂.</p> </div

Specificity of AM-1638 to GPR40 (FFAR1) <i>in vivo</i> and effect of the GLP-1R antagonist GLP-1(9–39)NH₂.

<p>An OGTT was performed in (A) wild type or (B) GPR40 null mice following a single oral dose of AM-1638 or sitagliptin. Glucose was dosed 1-hr post drug treatment. (C) Glucose AUC during OGTT. (D) GLP-1 secretion following a single oral dose of AM-1638 in wild type or GPR40 null mice. AM-1638 (60 mg/kg) was tested in an IPGTT in the presence or absence of the GLP-1R antagonist GLP-1(9–39)NH₂ (300 µg/kg) as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046300#s4" target="_blank">Materials and Methods</a> section. (E) Plasma glucose levels (F) Glucose AUC and (G) plasma insulin levels at the indicated timepoints during the experiment. Statistical significance compared to vehicle treatment is denoted by *(p<0.05), **(p<0.01), ***(p<0.001) and ****(p<0.0001), as determined by one-way or two-way ANOVA, and are color-coded to the treatment in the figure legends.</p

<i>In vitro</i> characterization of AM-1638 and AM-6226 and comparison to AMG 837.

<p>(A) Aequorin Ca²⁺ assay comparing AMG 837 to natural fatty acid ligands DHA, α-LNN and arachidonic acid. (B) Chemical structures of the key compounds synthesized during the medicinal chemistry effort that led to the discovery of AM-1638 and AM-6226. (C) Aequorin Ca²⁺ flux with key synthetic agonists and fatty acids. (D) Inositol phosphate assay with key synthetic agonists and fatty acids. (E–G) Plasmid titration experiments to examine agonist activity under conditions with reduced receptor levels, where either 5000 ng (E), 500 ng (F) or 50 ng (G) of GPR40 (FFAR1) expression plasmid was co-transfected with aequorin expression plasmids into CHO cells. (H) Competition binding experiment with ³H-AMG 837. (I) Competition binding experiment with ³H-AM-1638.</p

During an OGTT in NONcNZO10/LtJ type 2 diabetic mice, AM-1638 lowers blood glucose levels through an increase in insulin and incretin secretion.

<p>Vehicle (purple, n = 8) or 60 mg/kg AM-1638 (green, n = 8) was administered 1-hour prior to an oral glucose bolus. (A) Glucose levels at various timepoints. (B) Glucose AUC values. (C) Plasma insulin levels (D) GLP-1 levels and (E) GIP levels at baseline (-60 minutes) and 15 minutes after glucose challenge. Statistical significance compared to vehicle treatment is denoted by *(p<0.05), **(p<0.01), ***(p<0.001) and ****(p<0.0001), as determined by two-way ANOVA or student’s t-test.</p

Engagement of the enteroendocrine axis in HF/STZ type 2 diabetic mice by AM-1638.

<p>Following administration of a single dose of the indicated treatments at t = 0 minutes in HF/STZ mice, measurements of (A) GLP-1 (B) insulin (C) glucose and (D) GIP were taken at various timepoints. Statistical significance compared to vehicle treatment is denoted by *(p<0.05), **(p<0.01), ***(p<0.001) and ****(p<0.0001), as determined by one-way or two-way ANOVA, and are color-coded to the treatment in the figure legends.</p

Model depicting the dual mechanism-of-action of GPR40 (FFAR1) full agonists to lower blood glucose levels.

<p>GPR40 full agonists engage both the enteroendocrine axis as well as the pancreatic β-cell axis. These pathways both lead to augmentation of glucose stimulated insulin secretion on the pancreatic β-cell. Additionally, GLP-1 has multiple physiological and pharmacological roles, such as inhibition of glucagon secretion, that could further benefit type 2 diabetics. GPR40 partial agonists such as AMG 837 engage only the pancreatic pancreatic β-cell axis <i>in vivo</i>.</p

Activity of AM-1638 and AM-6226 in primary cells.

<p>(A) GSIS assay in mouse islets incubated with a dose response of compounds in the presence of 16.7 mM glucose. (B) Mouse islet perifusion assay. The glucose concentration was raised from 3 mM to 16.7 mM at t = 20 minutes and returned to 3 mM glucose at t = 30 minutes. 10 µM compound was perifused through the entire experiment. (C) GSIS assay with islets from wild type or GPR40 knock-out mice. (D) Inositol phosphate assay with dispersed human islet cells. (E) GSIS assay with human islets incubated with GPR40 agonists and 12.5 mM glucose. (F) GLP-1 secretion assay with fetal rat intestinal cells. (G) GIP-1 secretion assay with fetal rat intestinal cells. (H) Inositol phosphate accumulation assay using the mouse GLUTag enteroendocrine L-cell line. Statistical significance is denoted by *(p<0.05), **(p<0.01), ***(p<0.001) and ****(p<0.0001), as determined by one-way or two-way ANOVA, and are color-coded to treatment in the figure legends. For (A), (B), (F), (G), and (H) statistical comparisons were made to AMG 837 treatment. For (C) and (E) statistical comparisons were made to vehicle treatment.</p

Enhanced <i>in vivo</i> efficacy of AM-1638 compared to AMG 837 in HF/STZ type 2 diabetic mice.

<p>Drug treatment was administered 1-hour prior to an oral glucose bolus. (A) Glucose levels during an OGTT in high-fat fed, streptozotocin treated type 2 diabetic mice. (B) Glucose AUC values. (C) Change in plasma insulin levels from baseline during an OGTT (D) Insulin AUC (E) Unbound (free) plasma drug concentration in plasma 1-hour following drug dose, as determined by MS. Statistical significance compared to vehicle treatment is denoted by *(p<0.05), **(p<0.01), ***(p<0.001) and ****(p<0.0001), as determined by one-way or two-way ANOVA, and are color-coded to the treatment in the figure legends.</p

Trefoil Factor 3 (TFF3) Is Regulated by Food Intake, Improves Glucose Tolerance and Induces Mucinous Metaplasia

<div><p>Trefoil factor 3 (TFF3), also called intestinal trefoil factor or Itf, is a 59 amino acid peptide found as a homodimer predominantly along the gastrointestinal tract and in serum. TFF3 expression is elevated during gastrointestinal adenoma progression and has been shown to promote mucosal wound healing. Here we show that in contrast to other trefoil factor family members, TFF1 and TFF2, TFF3 is highly expressed in mouse duodenum, jejunum and ileum and that its expression is regulated by food intake. Overexpression of TFF3 using a recombinant adeno-associated virus (AAV) vector, or daily administration of recombinant TFF3 protein in vivo improved glucose tolerance in a diet-induced obesity mouse model. Body weight, fasting insulin, triglyceride, cholesterol and leptin levels were not affected by TFF3 treatment. Induction of mucinous metaplasia was observed in mice with AAV-mediated TFF3 overexpression, however, no such adverse histological effect was seen after the administration of recombinant TFF3 protein. Altogether these results suggest that the therapeutic potential of targeting TFF3 to treat T2D may be limited.</p></div