Male GDF15 were more prone to high fat diet-induced obesity.

<p>(A) Body weight of male mice. (B) Body weight of female mice. (C) Average daily food intake. (D) Body composition of male mice. n = 12–21 for male mice. n = 11–22 for female mice. Data are shown as mean±SEM. *p<0.05, ***p<0.001 between WT and KO by ANOVA.</p

Male GDF15 knockout DIO mice had higher glucose levels, insulin levels and worsened glucose tolerance.

<p>(A) 4hr fasting blood glucose levels of male mice. (B) 4hr fasting blood glucose levels of female mice. (C) 4hr fasting serum insulin levels of male mice. (D) 4hr fasting serum insulin levels of female mice. (E) Blood glucose levels of male mice during oral glucose tolerance test. (F) Blood glucose levels of female mice during oral glucose tolerance test. n = 12–21 for male mice. n = 11–22 for female mice. Data are shown as mean±SEM. *p<0.05, **p<0.01, ***p<0.001 between WT and KO by 2-Way ANOVA.</p

Metabolic rate and RER of female mice.

<p>(A) Oxygen consumption of female DIO mice: continuous 3-d recording and average value of each light cycle. (B) RER of female DIO mice: continuous 3-d recording and average value of each light cycle. (C) Heat production of female DIO mice: continuous 3-d recording and average value of each light cycle. n = 5–7. CLAMS recorded data are shown as mean. Analyzed average values are shown as mean±SEM.</p

Male GDF15 knockout DIO mice had lower metabolic rate than wildtype mice.

<p>(A) Oxygen consumption of male DIO mice: continuous 3-d recording and average value of each light cycle. (B) RER of male DIO mice: continuous 3-d recording and average value of each light cycle. (C) Heat production of male DIO mice: continuous 3-d recording and average value of each light cycle. n = 6. CLAMS recorded data are shown as mean. Analyzed average values are shown as mean±SEM. *p<0.05 between WT and KO by unpaired t-test.</p

Male GDF15 knockout DIO mice had lower locomotor activity.

<p>(A) X-axis activity of male DIO mice: continuous 3-d recording and average value of each light cycle. (B) Z-axis activity of male DIO mice: continuous 3-d recording and average value of each light cycle. (C) Continuous 3-d recording of food intake of male DIO mice. (D) Cumulative food intake of male DIO mice. n = 6. Raw CLAMS recorded data are shown as mean. Analyzed average values are shown as mean±SEM. *p<0.05, **p<0.01 between WT and KO by 2-way ANOVA.</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