The acute glucose lowering effect of specific GPR120 activation in mice is mainly driven by glucagon-like peptide 1

<div><p>The mechanism behind the glucose lowering effect occurring after specific activation of GPR120 is not completely understood. In this study, a potent and selective GPR120 agonist was developed and its pharmacological properties were compared with the previously described GPR120 agonist Metabolex-36. Effects of both compounds on signaling pathways and GLP-1 secretion were investigated <i>in vitro</i>. The acute glucose lowering effect was studied in lean wild-type and GPR120 <i>null</i> mice following oral or intravenous glucose tolerance tests. <i>In vitro</i>, in GPR120 overexpressing cells, both agonists signaled through Gα_q, Gα_s and the β-arrestin pathway. However, in mouse islets the signaling pathway was different since the agonists reduced cAMP production. The GPR120 agonists stimulated GLP-1 secretion both <i>in vitro</i> in STC-1 cells and <i>in vivo</i> following oral administration. <i>In vivo</i> GPR120 activation induced significant glucose lowering and increased insulin secretion after intravenous glucose administration in lean mice, while the agonists had no effect in GPR120 <i>null</i> mice. Exendin 9–39, a GLP-1 receptor antagonist, abolished the GPR120 induced effects on glucose and insulin following an intravenous glucose challenge. In conclusion, GLP-1 secretion is an important mechanism behind the acute glucose lowering effect following specific GPR120 activation.</p></div

Lipid accumulation and gene expression in primary macrophages from 8 weeks old <i>AdipoR2</i>^{<i>-/</i><i>-</i>}<i>ApoE</i>^<i>-/-</i> mice and <i>AdipoR2</i>^<i>+/+</i><i>ApoE</i>^<i>-/-</i> littermate controls.

<p>(A) Lipid accumulation in primary macrophages was analyzed after incubation with or without 50 μg/ml oxLDL for 24 h (Basal; <i>AdipoR2</i>^<i>+/+</i><i>ApoE</i>^<i>-/-</i><i> n</i> = 18, <i>AdipoR2</i>^{<i>-/</i><i>-</i>}<i>ApoE</i>^<i>-/-</i><i>n</i> = 15. OxLDL; <i>AdipoR2</i>^<i>+/+</i><i>ApoE</i>^<i>-/-</i><i> n</i> = 14, <i>AdipoR2</i>^{<i>-/</i><i>-</i>}<i>ApoE</i>^<i>-/-</i><i>n</i> = 13). Lipid accumulation is expressed as total oil red O area per cell. (B) CD36, (C) ABCA1, (D) IL-6, and (E) IL-10 mRNA expression in primary macrophages incubated with or without 50 μg/ml oxLDL for 24 h (Basal; <i>AdipoR2</i>^<i>+/+</i><i>ApoE</i>^<i>-/-</i><i> n</i> = 8, <i>AdipoR2</i>^{<i>-/</i><i>-</i>}<i>ApoE</i>^<i>-/-</i><i>n</i> = 6. OxLDL; <i>AdipoR2</i>^<i>+/+</i><i>ApoE</i>^<i>-/-</i><i> n</i> = 8, <i>AdipoR2</i>^{<i>-/</i><i>-</i>}<i>ApoE</i>^<i>-/-</i><i>n</i> = 5). Values are means ± SEM. *<i>P</i>< 0.05 for comparison between <i>AdipoR2</i>^{<i>-/</i><i>-</i>}ApoE^-/- and <i>AdipoR2</i>^<i>+/+</i>ApoE^-/- macrophages within each treatment group (Mann-Whitney <i>U</i> test). </p

Plaque area in the brachiocephalic artery of <i>AdipoR2</i>^{<i>-/</i><i>-</i>}ApoE^-/- mice and <i>AdipoR2</i>^<i>+/+</i><i>ApoE</i>^<i>-/-</i> littermate controls after 7 and 14 weeks on Lard diet.

<p>(A) Plaque area in the brachiocephalic artery measured by ultrasound at 15 and 22 weeks of age (<i>AdipoR2</i>^<i>+/+</i><i>ApoE</i>^<i>-/-</i><i>n</i> = 25, <i>AdipoR2</i>^{<i>-/</i><i>-</i>}<i>ApoE</i>^<i>-/-</i><i>n</i> = 14). (B) Plaque area in the brachiocephalic artery measured by histology at termination at 22 weeks of age (<i>AdipoR2</i>^<i>+/+</i><i>ApoE</i>^<i>-/-</i><i>n</i> = 24, <i>AdipoR2</i>^{<i>-/</i><i>-</i>}<i>ApoE</i>^<i>-/-</i><i>n</i> = 14). (C) Plaque area in the aortic arch measured by histology at termination at 22 weeks of age (<i>AdipoR2</i>^<i>+/+</i><i>ApoE</i>^<i>-/-</i><i>n</i> = 25, <i>AdipoR2</i>^{<i>-/</i><i>-</i>}<i>ApoE</i>^<i>-/-</i><i>n</i> = 11). (D) Plaque area (% of total vessel area) in the descending aorta measured by en face analysis at termination at 22 weeks of age (<i>AdipoR2</i>^<i>+/+</i><i>ApoE</i>^<i>-/-</i><i>n</i> = 22, <i>AdipoR2</i>^{<i>-/</i><i>-</i>}<i>ApoE</i>^<i>-/-</i><i>n</i> = 14). Miller´s elastin staining (E), Mac2 staining (F), and Picrosirius Red staining (G) of representative brachiocephalic artery plaques from <i>AdipoR2</i>^<i>+/+</i>ApoE^-/- mice (I-II) and <i>AdipoR2</i>^{<i>-/</i><i>-</i>}<i>ApoE</i>^<i>-/-</i> mice (III-IV). Values are means ± SEM. *<i>P</i>< 0.05, **<i>P</i>< 0.01 for comparison between <i>AdipoR2</i>^{<i>-/</i><i>-</i>}ApoE^-/- and <i>AdipoR2</i>^<i>+/+</i>ApoE^-/- mice (Mann-Whitney <i>U</i> test). </p

AdipoR2 protein expression in livers from <i>AdipoR2</i>^{<i>-/</i><i>-</i>}ApoE^-/- mice and <i>AdipoR2</i>^<i>+/+</i><i>ApoE</i>^<i>-/-</i> littermate controls.

<p>Absence of AdipoR2 protein in <i>AdipoR2</i>^{<i>-/</i><i>-</i>}<i>ApoE</i>^<i>-/-</i> mice was confirmed by western blot analysis of liver protein extractions using affinity purified AdipoR2 antibodies (<i>AdipoR2</i>^<i>+/+</i><i>ApoE</i>^<i>-/-</i><i>n</i> = 4, <i>AdipoR2</i>^{<i>-/</i><i>-</i>}<i>ApoE</i>^<i>-/-</i><i>n</i> = 4).</p

<i>In vitro</i> pharmacology of AZ13581837 and Metabolex-36.

<p><i>In vitro</i> pharmacology of AZ13581837 and Metabolex-36.</p

Plasma lipids in 22 weeks old <i>AdipoR2</i>^{<i>-/</i><i>-</i>}<i>ApoE</i>^<i>-/-</i> mice and <i>AdipoR2</i>^<i>+/+</i><i>ApoE</i>^<i>-/-</i> littermate controls after 14 weeks on Lard diet.

<p>(A) Cholesterol distribution profiles were measured by size exclusion HPLC on pooled plasma from 10 mice in each group. (B) Plasma triglycerides were measured in lipid extracts from plasma after separation by normal phase liquid chromatography, NPLC (<i>AdipoR2</i>^<i>+/+</i><i>ApoE</i>^<i>-/-</i><i>n</i> = 25, <i>AdipoR2</i>^{<i>-/</i><i>-</i>}<i>ApoE</i>^<i>-/-</i><i>n</i> = 14). Values are means ± SEM. *<i>P</i>< 0.05 (Mann-Whitney <i>U</i> test). </p

Exendin 9–39 blocked the AZ13581837 induced potentiation of insulin secretion in lean mice.

<p>Insulin levels following intravenous glucose challenge (<b>A</b>) and corresponding blood glucose (<b>C</b>), after administration of AZ13581837, exendin 9–39 or a co-administration of both, with corresponding calculations of AIR (<b>B</b>) and glucose elimination (<b>D</b>). The IVGTT data are from two independent experiments with n = 10 mice per group. Data are presented as mean ± SEM.***p<0.001 and *p<0.05 versus vehicle control.</p

Exendin 9–39 blocked the Metabolex-36 induced potentiation of insulin secretion in lean mice.

<p>Insulin levels following intravenous glucose challenge (<b>A</b>) and corresponding blood glucose (<b>C</b>), after administration of Metabolex-36, exendin 9–39 or a co-administration of both, with corresponding calculations of AIR (<b>B</b>) and glucose elimination (<b>D</b>). The IVGTT data are from two independent experiments with 6–7 mice per group. Data are presented as mean ± SEM.**p<0.01 and *p<0.05 versus vehicle control.</p

Metabolex-36 and AZ13581837 increased insulin secretion in IVGTT in lean mice.

<p>Insulin (<b>A</b>) and blood glucose (<b>C</b>) levels following an intravenous glucose challenge after oral administration of Metabolex-36 and AZ13581837 in lean female mice and corresponding AIR (<b>B</b>) and glucose elimination (<b>D</b>). Data represent six (Metabolex-36, n = 33, vehicle n = 34) and two (AZ13581837, n = 14) independent experiments and data are presented as mean ± SEM. Plasma levels of total GLP-1 (<b>E</b>) at time point was determined in separate experiments with n = 10 mice per group. ***p<0.001 and **p<0.01versus vehicle control.</p

Effect of Metabolex-36 and AZ13581837 on oral glucose tolerance in mice.

<p>Effect of Metabolex-36 (<b>A</b>) and AZ13581837 (<b>C</b>) on glucose response after an oral glucose challenge (2g/kg) in male mice and the corresponding unbound circulating concentrations of Metabolex-36 (<b>B</b>) and AZ13581837 (<b>D</b>) during the experiment. AZ13581837 and Metabolex-36 were given in different doses as indicated in the figures with n = 10 mice group and compared to vehicle treated mice (n = 12 mice per group). The EC₅₀ value for each GPR120 agonist assessed on mouse GPR120 using a DMR assay is indicated in figures. Blood glucose levels following oral glucose administration in GPR120 <i>null</i> mice (<b>E</b>) and wild type mice (<b>F</b>) were determined for vehicle (open squares) and Metabolex-36 (filled squares).</p