The aporphine alkaloid boldine induces adiponectin expression and regulation in 3T3-L1 cells

Abstract

Adiponectin is an adipokine secreted by differentiated adipocytes. Clinical studies suggest a negative correlation between oxidative stress and adiponectin levels in patients with metabolic syndrome or cardiovascular disease. Natural compounds that can prevent oxidative stress mediated inhibition of adiponectin may be potentially therapeutic. Boldine, an aporphine alkaloid abundant in the medicinal plant Peumus boldus, is a powerful antioxidant. The current study demonstrates the effects of boldine on the expression of adiponectin and its regulators, CCAAT/enhancer binding protein-alpha (C/EBPalpha) and peroxisome proliferator-activated receptor (PPAR)-gamma, in 3T3-L1 cells. Differentiated 3T3-L1 adipocytes were exposed to either hydrogen peroxide (H(2)O(2)) (100 microM) or tumor necrosis factor-alpha (TNFalpha) (1 ng/mL) for 24 hours in the presence or absence of increasing concentrations of boldine (5-100 microM). Quantitative polymerase chain reaction showed that both the oxidants decreased the mRNA levels of adiponectin, PPARgamma, and C/EBPalpha to half of the control levels. Boldine, at all concentrations, counteracted the inhibitory effect of H(2)O(2) or TNFalpha and increased the expression of adiponectin and its regulators. The effect of boldine on adiponectin expression was biphasic, with the lower concentrations (5-25 microM) having a larger inductive effect compared to higher concentrations (50-100 microM). Boldine treatment alone in the absence of H(2)O(2) or TNFalpha was also able to induce adiponectin at the inductive phase of adipogenesis. Peroxisome proliferator response element-luciferase promoter transactivity analysis showed that boldine interacts with the PPAR response element and could potentially modulate PPAR responsive genes. Our results indicate that boldine is able to modulate the expression of adiponectin and its regulators in 3T3-L1 cells and has the potential to be beneficial in obesity-related cardiovascular disease.

FIG. 1.

Effect of H₂O₂ and antioxidants on mRNA levels of (A) adiponectin and its transcription regulators, (B) PPARγ and (C) C/EBPα. Differentiated 3T3-L1 adipocytes were treated with H₂O₂ (100 μM) in the absence or presence of various antioxidants (α-tocopherol, NAC, and boldine) at 5–25 μM for 24 hours. After treatments, RT-qPCR was performed on isolated mRNA. The results are expressed as differences in fold change in antioxidant-treated cells compared to vehicle controls. Data are mean ± SEM values for three independent experiments performed in triplicate. *P ≤ .05, **P ≤ .01 versus control.

FIG. 2.

Effect of increasing concentrations of boldine on mRNA levels of (A) adiponectin and its transcription regulators, (B) PPARγ and (C) C/EBPα, in the presence of H₂O₂. Differentiated 3T3-L1 adipocytes were treated with H₂O₂ (100 μM) in the absence or presence of 5–100 μM boldine for 24 hours. After treatments, RT-qPCR was performed on isolated mRNA. The mRNA results are expressed as differences in fold change in antioxidant treated cells compared to vehicle controls. (D) The levels of secreted adiponectin protein were measured using Western blot analysis of the lyophilized cell supernatant from antioxidant-treated cells and are expressed as the percentage of their immunoblot intensity relative to vehicle controls. Data are mean ± SEM values for three independent experiments performed in triplicate. *P ≤ .05, **P ≤ .01 versus control.

FIG. 3.

Effect of increasing concentrations of boldine on mRNA levels of (A) adiponectin and its transcription regulators, (B) PPARγ and (C) C/EBPα, in the presence of TNFα. Differentiated 3T3-L1 adipocytes were treated with 1 ng/mL TNFα in the absence or presence of 5–100 μM boldine for 24 hours. After treatments, RT-qPCR was performed on isolated mRNA. The mRNA results are expressed as differences in fold change in antioxidant treated cells compared to vehicle controls. (D) The levels of secreted adiponectin protein were measured using Western blot analysis of the lyophilized cell supernatant from antioxidant-treated cells and are expressed as the percentage of their immunoblot intensity relative to vehicle controls. Data are mean ± SEM values for three independent experiments performed in triplicate. *P ≤ .05, **P ≤ .01 versus control.

FIG. 4.

Effect of treatment with increasing concentrations of boldine on adiponectin levels in the absence of H₂O₂: (A) mRNA levels for adiponectin and (B) Western blotting of secreted adiponectin protein. Differentiated 3T3-L1 adipocytes were exposed to increasing concentrations of boldine (5–100 μM) in the absence of any oxidants for 24 hours. After treatments, RT-qPCR was performed on isolated mRNA, and Western blotting was performed on the lyophilized cell supernatant. (C) Effect of boldine on adiponectin mRNA levels at various phases during the adipogenesis cascade. 3T3-L1 preadipocytes were exposed to 10 μM boldine at different phases during the adipogenesis process: day 0, day 2, day 4, and day 6. After treatments, RT-qPCR was performed on isolated mRNA. The mRNA results are expressed as differences in fold change in treated cells compared to untreated controls. Western blot results are expressed as percentage change in level of secreted adiponectin protein in treated cells compared to untreated controls. Data are mean ± SEM values for three independent experiments performed in triplicate. *P ≤ .05, ***P ≤ .005 versus control (CTRL).

FIG. 5.

Effect of boldine on PPRE-promoter activity. 3T3-L1 preadipocytes transfected with a PPRE-luciferase reporter construct was exposed to boldine (10 μM), the PPARγ agonist 15-PGJ2 (1 μM), or boldine (10 μM) + 15-PGJ2 (1 μM). PPRE-luciferase activity was measured as a change in chemiluminesence in a luminometer after a 24-hour treatment. The results are expressed as change in relative luminescence units (RLU) in treated cells compared to untreated controls. Data are mean ± SEM vales for five independent experiments performed in triplicate. *P ≤ .05 versus control.