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Piperine inhibits ABCA1 degradation and promotes cholesterol efflux from THP-1-derived macrophages

View Article: PubMed Central - PubMed

ABSTRACT

Scope: Increased macrophage cholesterol efflux (ChE) is considered to have anti-atherosclerotic effect counteracting cardiovascular disease. The principle pungent ingredient of the fruits of Piper nigrum, piperine, is identified in this study as a ChE inducer in THP-1-derived macrophages, and mechanisms underlying this effect are explored.

Methods and results: Without affecting cell viability, piperine concentration-dependently enhances ChE in THP-1-derived macrophages from 25 to 100 μM. The expression level of the key cholesterol transporter protein ATP-binding cassette transporter A1 (ABCA1) is significantly upregulated by piperine, as revealed by western blot analyses. However, two other ChE-mediating transporter proteins, ATP-binding cassette transporter G1 (ABCG1) and scavenger receptor class B member 1 (SR-B1), remain unaffected. Piperine exerts no influence on ABCA1 mRNA levels, but significantly inhibits the degradation of ABCA1, as evident by an increased half-life of the protein in the presence of cycloheximide. Furthermore, it is found that piperine likely interferes with the calpain-mediated ABCA1 degradation pathway and exhibits significant inhibition of calpain activity.

Conclusion: Our findings suggest that piperine promotes ChE in THP-1-derived macrophages by upregulation of ABCA1, which might be mediated by inhibition of calpain activity. This novel bioactivity makes the dietary constituent piperine a good candidate to be further explored for therapeutic or preventive applications in the context of atherosclerosis.

No MeSH data available.


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Expression of cholesterol transporter proteins in the absence and presence of piperine. (A–C) Differentiated THP-1-derived macrophages were treated with solvent vehicle control (DMSO; indicated as not treated with piperine or pioglitazone), piperine (PIP, 50 µM), or the PPARγ agonist pioglitazone (PIO, 10 µM) as positive control. After 24 h incubation, cells were lysed and 20 µg protein was resolved via SDS-PAGE. Immunodetection was performed with antibodies against the indicated proteins, ABCA1 (A), ABCG1 (B), and SR-B1 (C), and the protein bands were visualized by chemiluminescence detection. All values are mean ± SD (n = 4) versus solvent vehicle control, ***p < 0.001; n.s., no significance (one-way ANOVA/Bonferroni). (D) Differentiated THP-1 macrophages were incubated with 50 µM piperine (PIP) and 10 µM pioglitazone (PIO) for 24 h. Total RNA was extracted and ABCA1 mRNA expression levels were quantified by qRT-PCR. Data are mean ± S.D. (n = 5) versus solvent vehicle control (DMSO), **p < 0.01; ***p < 0.01; n.s., no significance (one-way ANOVA/Bonferroni.
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Figure 3: Expression of cholesterol transporter proteins in the absence and presence of piperine. (A–C) Differentiated THP-1-derived macrophages were treated with solvent vehicle control (DMSO; indicated as not treated with piperine or pioglitazone), piperine (PIP, 50 µM), or the PPARγ agonist pioglitazone (PIO, 10 µM) as positive control. After 24 h incubation, cells were lysed and 20 µg protein was resolved via SDS-PAGE. Immunodetection was performed with antibodies against the indicated proteins, ABCA1 (A), ABCG1 (B), and SR-B1 (C), and the protein bands were visualized by chemiluminescence detection. All values are mean ± SD (n = 4) versus solvent vehicle control, ***p < 0.001; n.s., no significance (one-way ANOVA/Bonferroni). (D) Differentiated THP-1 macrophages were incubated with 50 µM piperine (PIP) and 10 µM pioglitazone (PIO) for 24 h. Total RNA was extracted and ABCA1 mRNA expression levels were quantified by qRT-PCR. Data are mean ± S.D. (n = 5) versus solvent vehicle control (DMSO), **p < 0.01; ***p < 0.01; n.s., no significance (one-way ANOVA/Bonferroni.

Mentions: ABCA1, ABCG1, and SR-B1 are the most important transporter proteins for ChE in THP-1 macrophages. To investigate how piperine increases ChE, we first tested the expression levels of these three transporter proteins. Piperine significantly induced apo A1-mediated ChE in THP-1 macrophages (Fig. 2A) and it is known that ABCA1 exports intracellular cholesterol most efficiently to apo A1 [5]. Indeed, Fig. 3A shows that ABCA1 protein levels are significantly upregulated upon piperine (50 μM) treatment consolidating a significant role for this transporter in apo A1-mediated ChE. To determine whether piperine acts selective on ABCA1, we also examined the expression levels of ABCG1 and SR-B1 proteins in the presence of piperine. However, neither ABCG1 nor SR-B1 was affected upon piperine treatment (Fig. 3B and C). Pioglitazone, known to augment ABCA1 and ABCG1 protein levels in macrophages [29], was used as positive control.


Piperine inhibits ABCA1 degradation and promotes cholesterol efflux from THP-1-derived macrophages
Expression of cholesterol transporter proteins in the absence and presence of piperine. (A–C) Differentiated THP-1-derived macrophages were treated with solvent vehicle control (DMSO; indicated as not treated with piperine or pioglitazone), piperine (PIP, 50 µM), or the PPARγ agonist pioglitazone (PIO, 10 µM) as positive control. After 24 h incubation, cells were lysed and 20 µg protein was resolved via SDS-PAGE. Immunodetection was performed with antibodies against the indicated proteins, ABCA1 (A), ABCG1 (B), and SR-B1 (C), and the protein bands were visualized by chemiluminescence detection. All values are mean ± SD (n = 4) versus solvent vehicle control, ***p < 0.001; n.s., no significance (one-way ANOVA/Bonferroni). (D) Differentiated THP-1 macrophages were incubated with 50 µM piperine (PIP) and 10 µM pioglitazone (PIO) for 24 h. Total RNA was extracted and ABCA1 mRNA expression levels were quantified by qRT-PCR. Data are mean ± S.D. (n = 5) versus solvent vehicle control (DMSO), **p < 0.01; ***p < 0.01; n.s., no significance (one-way ANOVA/Bonferroni.
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Figure 3: Expression of cholesterol transporter proteins in the absence and presence of piperine. (A–C) Differentiated THP-1-derived macrophages were treated with solvent vehicle control (DMSO; indicated as not treated with piperine or pioglitazone), piperine (PIP, 50 µM), or the PPARγ agonist pioglitazone (PIO, 10 µM) as positive control. After 24 h incubation, cells were lysed and 20 µg protein was resolved via SDS-PAGE. Immunodetection was performed with antibodies against the indicated proteins, ABCA1 (A), ABCG1 (B), and SR-B1 (C), and the protein bands were visualized by chemiluminescence detection. All values are mean ± SD (n = 4) versus solvent vehicle control, ***p < 0.001; n.s., no significance (one-way ANOVA/Bonferroni). (D) Differentiated THP-1 macrophages were incubated with 50 µM piperine (PIP) and 10 µM pioglitazone (PIO) for 24 h. Total RNA was extracted and ABCA1 mRNA expression levels were quantified by qRT-PCR. Data are mean ± S.D. (n = 5) versus solvent vehicle control (DMSO), **p < 0.01; ***p < 0.01; n.s., no significance (one-way ANOVA/Bonferroni.
Mentions: ABCA1, ABCG1, and SR-B1 are the most important transporter proteins for ChE in THP-1 macrophages. To investigate how piperine increases ChE, we first tested the expression levels of these three transporter proteins. Piperine significantly induced apo A1-mediated ChE in THP-1 macrophages (Fig. 2A) and it is known that ABCA1 exports intracellular cholesterol most efficiently to apo A1 [5]. Indeed, Fig. 3A shows that ABCA1 protein levels are significantly upregulated upon piperine (50 μM) treatment consolidating a significant role for this transporter in apo A1-mediated ChE. To determine whether piperine acts selective on ABCA1, we also examined the expression levels of ABCG1 and SR-B1 proteins in the presence of piperine. However, neither ABCG1 nor SR-B1 was affected upon piperine treatment (Fig. 3B and C). Pioglitazone, known to augment ABCA1 and ABCG1 protein levels in macrophages [29], was used as positive control.

View Article: PubMed Central - PubMed

ABSTRACT

Scope: Increased macrophage cholesterol efflux (ChE) is considered to have anti-atherosclerotic effect counteracting cardiovascular disease. The principle pungent ingredient of the fruits of Piper nigrum, piperine, is identified in this study as a ChE inducer in THP-1-derived macrophages, and mechanisms underlying this effect are explored.

Methods and results: Without affecting cell viability, piperine concentration-dependently enhances ChE in THP-1-derived macrophages from 25 to 100 &mu;M. The expression level of the key cholesterol transporter protein ATP-binding cassette transporter A1 (ABCA1) is significantly upregulated by piperine, as revealed by western blot analyses. However, two other ChE-mediating transporter proteins, ATP-binding cassette transporter G1 (ABCG1) and scavenger receptor class B member 1 (SR-B1), remain unaffected. Piperine exerts no influence on ABCA1 mRNA levels, but significantly inhibits the degradation of ABCA1, as evident by an increased half-life of the protein in the presence of cycloheximide. Furthermore, it is found that piperine likely interferes with the calpain-mediated ABCA1 degradation pathway and exhibits significant inhibition of calpain activity.

Conclusion: Our findings suggest that piperine promotes ChE in THP-1-derived macrophages by upregulation of ABCA1, which might be mediated by inhibition of calpain activity. This novel bioactivity makes the dietary constituent piperine a good candidate to be further explored for therapeutic or preventive applications in the context of atherosclerosis.

No MeSH data available.


Related in: MedlinePlus