Glucose-Dependent Insulinotropic Polypeptide Prevents the Progression of Macrophage-Driven Atherosclerosis in Diabetic Apolipoprotein E-Null Mice

Aim: We recently reported that glucose-dependent insulinotropic polypeptide (GIP) prevents the development of atherosclerosis in apolipoprotein E-null (Apoe 2/2) mice. GIP receptors (GIPRs) are found to be severely down-regulated in diabetic animals. We examined whether GIP can exert anti-atherogenic effects in diabetes. Methods: Nondiabetic Apoe 2/2 mice, streptozotocin-induced diabetic Apoe 2/2 mice, and db/db mice were administered GIP (25 nmol/kg/day) or saline (vehicle) through osmotic mini-pumps for 4 weeks. The animals were assessed for aortic atherosclerosis and for oxidized low-density lipoprotein-induced foam cell formation in exudate peritoneal macrophages. Results: Diabetic Apoe 2/2 mice of 21 weeks of age exhibited more advanced atherosclerosis than nondiabetic Apoe 2/2 mice of the same age. GIP infusion in diabetic Apoe 2/2 mice increased plasma total GIP levels by 4-fold without improving plasma insulin, glucose, or lipid profiles. GIP infusion significantly suppressed macrophage-driven atherosclerotic lesions, but this effect was abolished by co-infusions with [Pro 3]GIP, a GIPR antagonist. Foam cell formation was stimulated by 3-fold in diabetic Apoe 2/2 mice compared with their nondiabetic counterparts, but this effect was halved by GIP infusion. GIP infusion also attenuated the foam cell formation in db/db mice. In vitro treatment with GIP (1 nM) reduced foam cell formation by 15 % in macrophages from diabetic Apoe 2/2 mice, and this attenuating effect was weaker than that attained by the same treatment of macrophages from nondiabetic counterparts (35%). While GIPR expression was reduced by onl

Glucagon-like Peptide-1 Suppresses the Proliferation and Migration of Vascular Smooth Muscle Cells: Implications for Preventive Effects on Atherosclerosis

Our group previously demonstrated the suppressive effect of glucagon-like peptide-1 (GLP-1) on macrophage-driven atherosclerosis in apolipoprotein E-deficient (apoE-/-) mice. In the present study we investigated the suppressive effect of GLP-1 on the atherogenic phenotype of vascular smooth muscle cells (VSMCs) in vivo using apoE-/- mice, and the proliferation and migration of human VSMCs in vitro. A 4-week infusion of GLP-1 in 17-week-old apoE-/- mice significantly reduced the proliferative VSMC phenotype stained with SMemb. Platelet-derived growth factor (PDGF) -BB significantly stimulated the proliferation of human aortic VSMCs by three fold. Both 0.1 and 1nmol/l GLP-1 significantly suppressed the PDGF-induced VSMC proliferation, and this suppressive effect was significantly abolished by the GLP-1 receptor antagonist exendin (9-39) (50nmol/l). The GLP-1 receptor agonists liraglutide (100nmol/l) and exendin-4 (100nmol/l) mimicked GLP-1, significantly suppressing PDGF-induced VSMC proliferation. PDGF-BB significantly stimulated the migration of human aortic VSMCs by 1.7 -fold, and this effect was significantly suppressed by 1nmol/l GLP-1. These findings suggest that GLP-1-related treatments may prevent the progression of atherosclerotic lesions by suppressing the proliferation and migration of VSMCs, which are characteristic features of atherosclerosis

<i>In vitro</i> suppressive effects of GIP on foam cell formation in exudate peritoneal macrophages from nondiabetic and diabetic <i>Apoe</i>^−/− mice.

<p>Exudate peritoneal macrophages were obtained from 6 nondiabetic <i>Apoe</i>^−/− mice (A) and 6 STZ-induced diabetic <i>Apoe</i>^−/− mice (B) by intraperitoneal injection of thioglycolate, and were incubated with or without GIP (1 nM) for 24 hours followed by addition of oxLDL (10 µg/ml) for 18 hours. Foam cell formation was evaluated by oxLDL-induced CE accumulation in macrophages. Assays were performed in duplicate. 1 fold = 15.6±1.5 nmol/mg cell protein (A) and 13.8±1.0 nmol/mg cell protein (B).</p

Foam cell formation in exudate peritoneal mouse macrophages.

<p><b>A</b>, 12 <i>Apoe</i>^−/− mice at 15 weeks of age were made diabetes by peritoneal injection of STZ (50 mg/kg/day) for 5 days and 6 <i>Apoe</i>^−/− mice were untreated. The 17-week-old diabetic <i>Apoe</i>^−/− mice were infused for 4 weeks with vehicle, GIP (25 nmol/kg/day), or GIP+[Pro³]GIP (both 25 nmol/kg/day) by osmotic mini-pumps. <b>B</b>, 6 <i>db/misty</i> mice and 6 <i>db/db</i> mice at 8 weeks of age were started to be infused with saline and 6 <i>db/db</i> mice were ifused with GIP (25 nmol/kg/day). Four weeks after infusions, exudate peritoneal macrophages were obtained from these mice by intraperitoneal injection of thioglycolate, and incubated with oxLDL (10 µg/ml) for 18 hours. Foam cell formation was evaluated by oxLDL-induced CE accumulation in macrophages. Assays were performed in duplicate or single. 1 fold = 17.5±1.0 nmol/mg cell protein (A) and 14.7±1.2 nmol/mg cell protein (B).</p

Suppressive effects of GIP on the progression of atherosclerotic lesions in diabetic <i>Apoe</i>^−/− mice.

<p>Thirty-five mice at 15 weeks of age were made diabetes by peritoneal injection of STZ (50 mg/kg/day) for 5 days and 14 mice were untreated. The 17-week-old diabetic <i>Apoe</i>^−/− mice were infused for 4 weeks with vehicle (control), GIP (25 nmol/kg/day), or GIP+[Pro³]GIP (both 25 nmol/kg/day) by osmotic mini-pumps. The aortic surface was stained with oil red O. Cross-sections of the aortic root were stained with oil red O or anti-MOMA-2 antibody. Hematoxylin was used for nuclear staining. The areas occupied by atherosclerotic lesions and by macrophage infiltration in the aortic wall were determined.</p

Expression of GIPR in pancreatic islets from <i>db/misty</i> and <i>db/db</i> mice.

<p>GIPR was stained with goat polyclonal anti-GIPR antibody. Hematoxylin was used for nuclear staining. Representative results are shown.</p

Expression of GIPR in exudate peritoneal macrophages from nondiabetic and diabetic <i>Apoe</i>^−/− mice.

<p>GIPR was stained with goat polyclonal anti-GIPR antibody followed by anti-goat Alexa Fluor 568. Phalloidin/DAPI staining shows F-actin cytoskeleton and nuclear morphology of mouse macrophages. These images were merged. Representative results are shown.</p

General characteristics and plasma measurements.

a<p> = vs. Nondiabetic,</p>b<p> = vs. Diabetic,</p>c<p> = Diabetic GIP at <i>P</i><0.001–0.05.</p

Surface atherosclerotic lesions of the aorta in 21-week-old nondiabetic <i>Apoe</i>^{−<b>/</b>−} mice treated without/with vildagliptin infused with incretin receptor blockers.

<p>During the 4-week period of vildagliptin administration, the <i>Apoe</i>^−/− mice (17-weeks-old at the outset) were respectively infused with saline (vehicle), GLP-1 receptor blocker (Ex-9), the GIPR blocker (Pro³), and Ex-9+Pro³ by osmotic mini-pump. The entire aorta was stained with Oil Red O.</p

The atherosclerotic area and the macrophage infiltration area in the aortic root in 21-week-old nondiabetic <i>Apoe</i>^{−<b>/</b>−} mice treated without/with vildagliptin infused with incretin receptor blockers.

<p>The cross-sections of the aortic root were stained with Oil Red O. The area of macrophage infiltration was stained with MOMA-2.</p