1. Differential effects of GLP-1 receptor agonist on foam cell formation in monocytes between non-obese and obese subjects 
Masashi Tanaka, Yoshiyuki Matsuo, Hajime Yamakage, Shinya Masuda, Yuko Terada, Kazuya Muranaka, Hiromichi Wada, Koji Hasegawa, Akira Shimatsu, Noriko Satoh-Asahara 
Metabolism - Clinical and Experimental 
Volume 65, Issue 2, Pages 1-11 (February 2016)
DOI: /j.metabol
Copyright © 2015 Elsevier Inc. Terms and Conditions

2. Fig. 1
Foam cell formation and proinflammatory cytokine production were inhibited by exendin-4 in ox-LDL-stimulated monocytes from control subjects but not in those from obese patients. A: Lipid content in monocytes from control subjects and obese patients. Freshly isolated monocytes were stained with BODIPY, followed by analysis with a flow cytometer. BODIPY-stained MFI is shown. B: Mitochondrial membrane potential in monocytes from control subjects and obese patients. Freshly isolated monocytes were stained with 250nmol/L MitoTracker Red CMXRos, followed by analysis with a flow cytometer. MitoTracker Red CMXRos-derived MFI is shown. C: Cell surface expression levels of GLP-1R on monocytes freshly isolated from control subjects and obese patients. The shaded histograms show isotype-matched control Ab (IgG2B)-staining, and the solid line histograms show specific staining. The right graph shows the MFI of GLP-1R+ monocytes from control subjects and obese patients. D: Effects of exendin-4 and Torin1 on foam cell formation of ox-LDL-stimulated monocytes from control subjects. Monocytes were stimulated with vehicle control or 50μg/mL ox-LDL for 24h in the presence or absence of 10nmol/L exendin-4 or 100nmol/L Torin1, followed by incubation with or without 5μg/mL BODIPY at 37°C for 30min. Then, the cells were analyzed by flow cytometry. Changes in the cellular neutral lipid content were assessed by comparing the mean fluorescent intensity (MFI) of BODIPY-stained cells with that of non-stained cells; the MFI of non-stained cells was subtracted from that of BODIPY-stained cells. The changes of the MFI are shown relative to the vehicle control. E: Effects of exendin-4 and Torin1 on foam cell formation of ox-LDL-stimulated monocytes from obese patients. The panel shows the relative changes in the lipid content, indicated as the BODIPY-derived MFI, of monocytes with or without 50μg/mL ox-LDL stimulation for 24h in the presence or absence of 10nmol/L exendin-4 or 100nmol/L Torin1. F: Effects of exendin-4 on proinflammatory cytokine production in monocytes from control subjects and obese patients. Monocytes were stimulated as indicated for 24h, and the amount of cytokines in the culture supernatant was quantified using Cytometric Bead Array. Data shown are representative of at least two independent experiments performed in triplicate. *P<0.05; **P<0.01.
Metabolism - Clinical and Experimental  , 1-11DOI: ( /j.metabol )
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3. Fig. 2
Exendin-4 induces indicators of autophagy in monocytes from control subjects but not in those from obese patients. A: Monocytes from control subjects were stimulated with or without 50μg/mL ox-LDL in the presence or absence of 10nmol/L exendin-4 for 12h. The total cell lysates were separated by SDS-PAGE, and the amounts of LC3-I, LC3-II, Beclin 1, and β-actin were analyzed. The graphs show densitometric analysis of the proteins indicated. B: Monocytes from control subjects were stimulated with or without 50μg/mL ox-LDL in the presence or absence of 100nmol/L Torin1 for 3h. The amounts of LC3-I, LC3-II, Beclin 1, and β-actin in total cell lysates were analyzed by Western blot. The graphs show densitometric analysis of the proteins indicated. C: Monocytes from obese patients were stimulated with or without 50μg/mL ox-LDL in the presence or absence of 10nmol/L exendin-4 for 12h, followed by Western blot. The graphs show densitometric analysis of the proteins indicated. D: Monocytes from obese patients were stimulated with or without 50μg/mL ox-LDL in the presence or absence of 100nmol/L Torin1 for 3h, followed by Western blot. The graphs show densitometric analysis of the proteins indicated. Data shown are representative of at least three independent experiments. *P<0.05; **P<0.01.
Metabolism - Clinical and Experimental  , 1-11DOI: ( /j.metabol )
Copyright © 2015 Elsevier Inc. Terms and Conditions

4. Fig. 3
The exendin-4-stimulated cAMP/PKA pathway is involved in regulating foam cell formation of human monocytes. A: The panel shows relative changes in the lipid content obtained by BODIPY staining as in Fig. 1A in monocytes from control subjects with or without 50μg/mL ox-LDL stimulation for 24h. It also shows the effects of MDL-12330A (5μmol/L), a specific adenylate cyclase inhibitor, or PKI14-22 (10μmol/L), a PKA inhibitor, on lipid accumulation in ox-LDL-stimulated monocytes from control subjects in the presence or absence of 10nmol/L exendin-4. The intensity of the BODIPY-stained MFI on vehicle treatment is set at 1.0, and relative changes are shown. B: The panel shows relative changes in the lipid content of monocytes from obese patients with or without 50μg/mL ox-LDL stimulation for 24h. It also shows the effects of MDL-12330A (5μmol/L) or PKI14-22 (10μmol/L) on lipid accumulation in ox-LDL-stimulated monocytes from obese patients in the presence or absence of 10nmol/L exendin-4. The value of vehicle treatment is set at 1.0, and relative changes are shown. Data shown are representative of at least two independent experiments performed in triplicate. *P<0.05; **P<0.01.
Metabolism - Clinical and Experimental  , 1-11DOI: ( /j.metabol )
Copyright © 2015 Elsevier Inc. Terms and Conditions

5. Fig. 4
Exendin-4 inhibited foam cell formation in parallel with induction of indicators of autophagy in THP-1 macrophages. A: Effects of exendin-4 and Torin1 on lipid accumulation in ox-LDL-stimulated THP-1 macrophages. THP-1 macrophages were stimulated with vehicle control or 50μg/mL ox-LDL for 24h in the presence or absence of exendin-4 or Torin1, followed by incubation with or without 5μg/mL BODIPY at 37°C for 30min. Then, the cells were analyzed by flow cytometry. Changes in the cellular neutral lipid content were assessed by comparing the mean fluorescent intensity (MFI) of BODIPY-stained cells with that of non-stained cells; the MFI of non-stained cells was subtracted from that of BODIPY-stained cells. The changes of the MFI are shown relative to the vehicle control. B: Effects of exendin-4 on IL-10 expression in THP-1 macrophages. THP-1 macrophages were stimulated with vehicle control or 50μg/mL ox-LDL for 6h in the presence of vehicle control or 250nmol/L exendin-4, followed by total RNA isolation. The mRNA expression level of IL-10 was determined by quantitative RT-PCR. Relative expression is displayed as the expression level in THP-1 macrophages without the addition of ox-LDL and exendin-4 set at 1.0. C: Effects of exendin-4 on autophagy induction. Left panels show Western blot of THP-1 macrophages stimulated with vehicle control or 50μg/mL ox-LDL in the presence of 250nmol/L exendin-4 for 6h or 250nmol/L Torin1 for 1h or vehicle control. Right panels show densitometric quantification of Western blot. Data shown are representative of at least three independent experiments performed in triplicate. *P<0.05; **P<0.01.
Metabolism - Clinical and Experimental  , 1-11DOI: ( /j.metabol )
Copyright © 2015 Elsevier Inc. Terms and Conditions

6. Fig. 5
Effects of Ex-4 on human monocytes. Ex-4 induces autophagy and suppresses foam cell formation and inflammation in monocytes from control subjects. The GLP-1R-mediated-suppression of foam cell formation and inflammation is dysregulated in monocytes from obese patients.
Metabolism - Clinical and Experimental  , 1-11DOI: ( /j.metabol )
Copyright © 2015 Elsevier Inc. Terms and Conditions