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

Characteristics and laboratory data from three groups of diabetic <i>db/db</i> mice with or without ipragliflozin, and wild-type mice that received vehicle.

<p>Non-diabetic wild-type mice that received vehicle, diabetic <i>db/db</i> mice that received vehicle, diabetic <i>db/db</i> mice that received ipragliflozin were measured. The values show mean ± SEM. Ipragliflozin, sodium glucose co-transporter 2 inhibitor.</p><p>One-way ANOVA followed by Tukey test:</p><p>^a,<i>p</i> < 0.05 vs. wild-type mice that received vehicle;</p><p>^b,<i>p</i> < 0.05 vs. diabetic <i>db/db</i> mice that received ipragliflozin.</p><p>Characteristics and laboratory data from three groups of diabetic <i>db/db</i> mice with or without ipragliflozin, and wild-type mice that received vehicle.</p

Foam cell formation in exudate peritoneal macrophages obtained from non-diabetic and diabetic <i>Apoe</i>^{<i>−/−</i>} mice (A), and diabetic <i>db/db</i> mice (B).

<p>Four days after an intraperitoneal injection of thioglycolate, the exudated peritoneal cells were isolated from the treated non-diabetic and diabetic <i>Apoe</i>^{<i>−/−</i>} mice at 21 weeks of age (Fig 3A), or from the diabetic <i>db/db</i> mice at 13 weeks of age (Fig 3B). Adherent macrophages were incubated for 18 hours with the RPMI-1640 medium containing 10μg/mL oxidized low-density lipoprotein (LDL) in the presence of 0.1 mmoL [³H]olate that was conjugated with bovine serum albumin. The cellular lipids were extracted and the radioactivity of the cholesterol [³H]olate was determined with thin-layer chromatography. Foam cell formation was expressed as cholesteryl ester (CE) accumulation. The values show mean ± SEM. One-way ANOVA followed by Tukey test: ^‡<i>p</i> < 0.001, ^§<i>p</i> < 0.0001.</p

Correlation between foam cell formation and glycemic control in diabetic <i>Apoe</i>^{<i>−/−</i>} mice (A) or diabetic <i>db/db</i> mice (B).

<p>Fig 5A shows the correlation between foam cell formation and HbA1c in non-diabetic and diabetic <i>Apoe</i>^{<i>−/−</i>} mice. Diabetic mice that received vehicle (n = 10); r = 0.77, <i>p</i> < 0.01. Diabetic mice that received dapagliflozin (n = 5); r = 0.98, <i>p</i> < 0.005. Combined diabetic mice (n = 15); r = 0.91, <i>p</i> < 0.0001. Fig 5B shows the correlation between foam cell formation and HbA1c in diabetic <i>db/db</i> mice and wild-type (C57/BL6) mice. Diabetic mice that received vehicle (n = 18); r = 0.71, <i>p</i> < 0.001. Diabetic mice that received ipragliflozin (n = 13); r = 0.87, <i>p</i> < 0.0001. Combined diabetic mice (n = 31); r = 0.88, <i>p</i> < 0.0001. <i>r</i> values indicate Pearson correlation coefficients. Pearson’s correlation test, <i>p</i> < 0.05.</p

Suppressive effects of dapagliflozine administration against the development of aortic atherosclerotic lesions in non-diabetic and diabetic <i>Apoe</i>^{<i>−/−</i>} mice.

<p>Twenty-two mice at 15 weeks of age were made diabetic with peritoneal injections of STZ (50 mg/kg/day) for 5 consecutive days and twenty mice were treated with saline. The 17-week-old non-diabetic and diabetic <i>Apoe</i>^{<i>−/−</i>} mice were orally given SGLT2i (dapagliflozin) or vehicle for 4 weeks, starting from 17 weeks of age. Representative atherosclerotic lesions in the aortic surface stained with oil red O (a-d) and measured (m). Yellow arrows show notable atherosclerotic lesions. In the aortic root, the atheromatous plaques and monocyte/macrophage accumulations were stained with Oil red O (e-h) or anti-MOMA2 antibody (i-l). Black arrows show notable atheromatous plaques. The severity of atheromatous plaques (n) and degree of monocyte/macrophage accumulation (o) were evaluated. The data are expressed as mean ± SEM. One way ANOVA followed by Tukey test: ^†<i>p</i> < 0.01, ^‡<i>p</i> < 0.001, ^§<i>p</i> < 0.0001.</p