Glucose-Dependent Insulinotropic Polypeptide Inhibits AGE-Induced NADPH Oxidase-Derived Oxidative Stress Generation and Foam Cell Formation in Macrophages Partly via AMPK Activation

Abstract

Glucose-dependent insulinotropic polypeptide (GIP) of the incretin group has been shown to exert pleiotropic actions. There is growing evidence that advanced glycation end products (AGEs), senescent macromolecules formed at an accelerated rate under chronic hyperglycemic conditions, play a role in the pathogenesis of atherosclerotic cardiovascular disease in diabetes. However, whether and how GIP could inhibit the AGE-induced foam cell formation of macrophages, an initial step of atherosclerosis remains to be elucidated. In this study, we address these issues. We found that AGEs increased oxidized low-density-lipoprotein uptake into reactive oxygen species (ROS) generation and Cdk5 and CD36 gene expressions in human U937 macrophages, all of which were significantly blocked by [D-Ala²]GIP(1-42) or an inhibitor of NADPH oxidase activity. An inhibitor of AMP-activated protein kinase (AMPK) attenuated all of the beneficial effects of [D-Ala²]GIP(1-42) on AGE-exposed U937 macrophages, whereas an activator of AMPK mimicked the effects of [D-Ala²]GIP(1-42) on foam cell formation, ROS generation, and Cdk5 and CD36 gene expressions in macrophages. The present study suggests that [D-Ala²]GIP(1-42) could inhibit the AGE-RAGE-induced, NADPH oxidase-derived oxidative stress generation in U937 macrophages via AMPK activation and subsequently suppress macrophage foam cell formation by reducing the Cdk5-CD36 pathway.

Keywords: AGEs; AMPK; CD36; Cdk5; GIP; NADPH oxidase.

Figure 1

Melt curve of gene expression of Gipr (A) and Rage (B) in human U937 cells.

Figure 2

Effects of [D-Ala²]GIP(1–42), dorsomorphin, AICAR, and DPI on Dil-ox-LDL uptake into, and ROS generation in, AGE-exposed U937 macrophages. U937 cells were treated with 100 μg/mL AGE-BSA or 100 μg/mL non-glycated control BSA in the presence or absence of 1 nmol/L [D-Ala²]GIP(1–42), 50 nmol/L dorsomorphin, 500 μmol/L AICAR, or 50 nmol/L DPI in Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 10% fetal bovine serum (FBS) at 37 °C for 24 h. Then, the adherent cells were incubated with 10 μg/mL Dil-ox-LDL for 18 h and further treated with a 10 μmol/L DCFH-DA probe for another 15 min. The fluorescence intensity of Dil-ox-LDL and DCF were measured. (A–R) Representative images of immunofluorescence staining. Dil-ox-LDL-positive cells were stained in red, and ROS-generating cells are represented in green. Scale bars show 20 μm. (S–U) Quantitative data of fluorescence intensity for Dil-ox-LDL and DCF were normalized for each control value with BSA-treated cells, respectively. The correlation of fluorescence intensity between Dil-ox-LDL and DCF was used by Pearson’s correlation test. Number = 10 for each group. Values are shown as mean ± standard deviation. ★★★ p < 0.005. (U) Blue circles for BSA-treated cells, red squares for AGE-treated cells, and green triangles for AGE-treated cells with [D-Ala²]GIP(1–42).

Figure 3

Effects of [D-Ala²]GIP(1–42), dorsomorphin, AICAR, and DPI on Cdk5 and CD36 gene expression levels in AGE-exposed U937 macrophages. Gene expression levels of Cdk5 (A) or CD36 (B) and their correlation (C). The correlation of Cdk5 with CD36 gene expression levels was determined by Pearson’s correlation test. Total RNAs were transcribed and amplified by quantitative real-time RT-PCR. The levels of the target gene were initialized by the value of GAPDH mRNA-derived signals and then relatively calculated by the controls with BSA. Number = 10 for each group. Values are presented as mean ± standard deviation. ★★★ p < 0.005. (C) Blue circles for BSA-treated cells, red squares for AGE-treated cells, and green triangles for AGE-treated cells with [D-Ala²]GIP(1–42).

Figure 4

Correlation of intracellular ROS generation with Cdk5 (A) or CD36 (B) mRNA levels and the association of CD36 gene expression levels with Dil-ox-LDL uptake (C) into U937 cells. The correlation was determined by Pearson’s correlation test. Number = 10 for each group. Blue circles for BSA-treated cells, red squares for AGE-treated cells, and green triangles for AGE-treated cells with [D-Ala²]GIP(1–42).

Figure 5

Possible mechanisms of the inhibitory effect of [D-Ala²]GIP(1–42) on foam cell formation of macrophages. AGEs, advanced glycation end products; RAGE, receptor for AGE; GIPR, receptor of glucose-dependent insulinotropic polypeptide; AMPK, AMP-activated protein kinase; Cdk5, cyclin-dependent kinase 5; CE, cholesterol ester; DPI, diphenylene iodonium; AICAR, 5-aminoimidazole-4-carboxamide1-B-D-ribofuranoside; and ox-LDL, oxidized low-density lipoprotein.