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 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

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

<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 Ucn1 on foam cell formation and related protein expression in human monocyte-derived macrophages.

<p>Human monocytes were incubated for 7 days with RPMI-1640 supplemented with 10% human serum and the indicated concentrations of Ucn1, followed by a 19 h-incubation with 50 µg/ml oxLDL in the presence of 0.1 mmol/l [³H]oleate. Intracellular CE accumulation was determined from the radioactivity of cholesterol-[³H]oleate. Otherwise, before the addition of oxLDL, cells were harvested and subjected to immunoblotting analyses for CD36, ACAT1, or ABCA1. β-Actin served as a loading control. Data are expressed as means ± SEM from 4–6 independent experiments with monocytes from 4–6 different donors. Baseline (1 fold) = 8.4±2.2 nmol/mg cell protein. *<i>P</i><0.05, <b>^†</b><i>P</i><0.001 vs. 0 nmol/l of Ucn1.</p

Primer Sequences Used for RT-PCR.

<p>IL6 = interleukin-6, MCP1 = monocyte chemoattractant protein-1, ICAM1 = intercellular adhesion molecule-1, GAPDH = glyceraldehyde-3-dehydrogenase.</p><p>Primer Sequences Used for RT-PCR.</p

Characteristics and laboratory data in 21-week-old <i>Apoe</i>^−/− mice.

<p>Mean ± SEM.</p><p>Characteristics and laboratory data in 21-week-old <i>Apoe</i>^−/− mice.</p

Stimulatory effects of Ucn1 on MMP2 and MMP9 activities in human VSMCs.

<p>HASMCs were incubated for 24 h in serum-free conditioning medium with the indicated concentrations of Ucn1. In the culture supernatant, MMP2 and MMP9 activities were determined by the zymography assay with 0.1% gelatin as a substrate in a 10% SDS-PAGE. Data are expressed as means ± SEM from 5 independent experiments. *<i>P</i><0.005, <b>^†</b><i>P</i><0.001, <b>^‡</b><i>P</i><0.05 vs. 0 nmol/l of Ucn1. M = MMP marker, S = sample.</p