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TNF-alpha and IL-6 inhibit apolipoprotein A-IV production induced by linoleic acid in human intestinal Caco2 cells.

Li X, Xu M, Liu M, Ji Y, Li Z - J Inflamm (Lond) (2015)

Bottom Line: LA stimulated gene and protein expression of apoA-IV in a dose and time dependent manner.Pre-treatment with the cytokines for 72 h significantly inhibited the increased expression of apoA-IV gene and protein induced by LA.Our data indicate that the pro-inflammatory cytokines may be responsible for the reduced apoA-IV production in response to fat feeding.

View Article: PubMed Central - PubMed

Affiliation: National Local Joint Engineering Research Center of Biodiagnostics and Biotherapy, The Second Affiliated Hospital of Medical College, Xi'an Jiaotong University, Xi'an, 710004 China ; Department of Pathology and Laboratory Medicine, University of Cincinnati, Cincinnati, 45237-0507 USA.

ABSTRACT

Background: Apolipoprotein A-IV (apoA-IV) is a protein mainly synthesized by enterocytes in the intestine. Its gene expression is suppressed during fasting and stimulated during active fat absorption. Chronic feeding of a high-fat (HF) diet abolishes the differential expression between fasting and fat-feeding and therefore may contribute to diet-induced obesity since apoA-IV is a potent satiety factor. It is well established that the circulating pro-inflammatory cytokines TNF-α and IL-6 are increased by HF feeding.

Methods: To determine whether pro-inflammatory cytokines are involved in the diminished response of apoA-IV gene expression to fat-feeding, different concentrations of linoleic acid (LA), an important dietary fatty acid, was used to stimulate apoA-IV expression in human intestinal Caco2 cells. Cells were pre-treated with or without human recombinant TNF-α, IL-6 or their combination before the addition of LA. Real-time PCR and ELISA were used to detect and quantify RNA transcripts and proteins of apoA-IV and the cytokines.

Results: LA stimulated gene and protein expression of apoA-IV in a dose and time dependent manner. Pre-treatment with the cytokines for 72 h significantly inhibited the increased expression of apoA-IV gene and protein induced by LA. Furthermore, the cytokines, especially TNF-α, also positively up-regulate the cytokine themselves in Caco2 cells.

Conclusions: Our data indicate that the pro-inflammatory cytokines may be responsible for the reduced apoA-IV production in response to fat feeding. Because of apoA-IV's role in satiety, we propose the inhibitory effect of circulating pro-inflammatory cytokines on apoA-IV production contributes to diet-induced obesity.

No MeSH data available.


Related in: MedlinePlus

Effects of cytokines onapoA-IVandapoC-IIIgene expression. (A) Effects of LA and cytokines on apoA-IV gene expression. Differentiated Caco2 cells were pre-treated with or without r-h-TNF-α (20 ng/ml) or r-h-IL-6 (20 ng/ml) or the combination of these two cytokines for 72 h before changing into medium with or without 1 mM LA for 24 h. (B) Effects of LA and cytokines on apoA-IV protein secretion. Differentiated Caco2 cells were pre-treated with or without r-h-TNF-α (20 ng/ml) or r-h-IL-6 (20 ng/ml) or the combination of these two cytokines for 72 h before changing into medium with or without 0.25 or 0.5 mM LA for 24 h. The apoA-IV proteins in culture medium secreted from the cells were measured by ELISA. (C) Effects of LA and cytokines on apoC-III expression. The samples were processed at the same ways described at (A). The mRNA levels of both apoA-IV and apoC-III were measured by real time RT-PCR. * P < 0.05, ** P < 0.01, *** P < 0.001 vs. vehicle control; and ##P < 0.01 ###P < 0.001 vs. LA.
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Fig2: Effects of cytokines onapoA-IVandapoC-IIIgene expression. (A) Effects of LA and cytokines on apoA-IV gene expression. Differentiated Caco2 cells were pre-treated with or without r-h-TNF-α (20 ng/ml) or r-h-IL-6 (20 ng/ml) or the combination of these two cytokines for 72 h before changing into medium with or without 1 mM LA for 24 h. (B) Effects of LA and cytokines on apoA-IV protein secretion. Differentiated Caco2 cells were pre-treated with or without r-h-TNF-α (20 ng/ml) or r-h-IL-6 (20 ng/ml) or the combination of these two cytokines for 72 h before changing into medium with or without 0.25 or 0.5 mM LA for 24 h. The apoA-IV proteins in culture medium secreted from the cells were measured by ELISA. (C) Effects of LA and cytokines on apoC-III expression. The samples were processed at the same ways described at (A). The mRNA levels of both apoA-IV and apoC-III were measured by real time RT-PCR. * P < 0.05, ** P < 0.01, *** P < 0.001 vs. vehicle control; and ##P < 0.01 ###P < 0.001 vs. LA.

Mentions: It has been reported that cytokine IL-6 and TNF-α reduced apoA-IV mRNA levels in pig hepatocytes [17]. To determine whether long time exposure to TNF-α and IL-6 also affects apoA-IV gene expression induced by LA in Caco2 cells, the differentiated Caco2 cells were pre-treated with r-h-TNFα and r-h-IL6 for 72 h, followed by incubation with or without 1 mM LA for 24 h. As shown in Figure 2A, the cytokines did not affect apoA-IV mRNA in the cells without LA treatment. However, pre-treatment with the combination of these two cytokines significantly attenuated LA-induced increase of mRNA levels (Figure 2A) and apoA-IV protein secretion (Figure 2B). To further determine which cytokine, TNF-α or IL-6, exerts the inhibitory effect, we pre-incubated the cells with either TNF-α or IL-6, respectively, followed by the LA treatment. We found that TNF-α significantly attenuated the increased levels of both apoA-IV mRNA (Figure 2A) and protein (Figure 2B) induced by LA, whereas IL-6 had no such effect on apoA-IV mRNA level (Figure 2A), but significantly attenuated apoA-IV protein level induced by 0.25 mM LA (Figure 2B). Furthermore, TNF-α and IL-6 themselves decreased apoA-IV protein levels (Figure 2B), but not apoA-IV mRNA levels (Figure 2A). These data indicate that both TNF-α and IL-6 inhibit the stimulatory effect of LA on apoA-IV production.Figure 2


TNF-alpha and IL-6 inhibit apolipoprotein A-IV production induced by linoleic acid in human intestinal Caco2 cells.

Li X, Xu M, Liu M, Ji Y, Li Z - J Inflamm (Lond) (2015)

Effects of cytokines onapoA-IVandapoC-IIIgene expression. (A) Effects of LA and cytokines on apoA-IV gene expression. Differentiated Caco2 cells were pre-treated with or without r-h-TNF-α (20 ng/ml) or r-h-IL-6 (20 ng/ml) or the combination of these two cytokines for 72 h before changing into medium with or without 1 mM LA for 24 h. (B) Effects of LA and cytokines on apoA-IV protein secretion. Differentiated Caco2 cells were pre-treated with or without r-h-TNF-α (20 ng/ml) or r-h-IL-6 (20 ng/ml) or the combination of these two cytokines for 72 h before changing into medium with or without 0.25 or 0.5 mM LA for 24 h. The apoA-IV proteins in culture medium secreted from the cells were measured by ELISA. (C) Effects of LA and cytokines on apoC-III expression. The samples were processed at the same ways described at (A). The mRNA levels of both apoA-IV and apoC-III were measured by real time RT-PCR. * P < 0.05, ** P < 0.01, *** P < 0.001 vs. vehicle control; and ##P < 0.01 ###P < 0.001 vs. LA.
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Related In: Results  -  Collection

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Fig2: Effects of cytokines onapoA-IVandapoC-IIIgene expression. (A) Effects of LA and cytokines on apoA-IV gene expression. Differentiated Caco2 cells were pre-treated with or without r-h-TNF-α (20 ng/ml) or r-h-IL-6 (20 ng/ml) or the combination of these two cytokines for 72 h before changing into medium with or without 1 mM LA for 24 h. (B) Effects of LA and cytokines on apoA-IV protein secretion. Differentiated Caco2 cells were pre-treated with or without r-h-TNF-α (20 ng/ml) or r-h-IL-6 (20 ng/ml) or the combination of these two cytokines for 72 h before changing into medium with or without 0.25 or 0.5 mM LA for 24 h. The apoA-IV proteins in culture medium secreted from the cells were measured by ELISA. (C) Effects of LA and cytokines on apoC-III expression. The samples were processed at the same ways described at (A). The mRNA levels of both apoA-IV and apoC-III were measured by real time RT-PCR. * P < 0.05, ** P < 0.01, *** P < 0.001 vs. vehicle control; and ##P < 0.01 ###P < 0.001 vs. LA.
Mentions: It has been reported that cytokine IL-6 and TNF-α reduced apoA-IV mRNA levels in pig hepatocytes [17]. To determine whether long time exposure to TNF-α and IL-6 also affects apoA-IV gene expression induced by LA in Caco2 cells, the differentiated Caco2 cells were pre-treated with r-h-TNFα and r-h-IL6 for 72 h, followed by incubation with or without 1 mM LA for 24 h. As shown in Figure 2A, the cytokines did not affect apoA-IV mRNA in the cells without LA treatment. However, pre-treatment with the combination of these two cytokines significantly attenuated LA-induced increase of mRNA levels (Figure 2A) and apoA-IV protein secretion (Figure 2B). To further determine which cytokine, TNF-α or IL-6, exerts the inhibitory effect, we pre-incubated the cells with either TNF-α or IL-6, respectively, followed by the LA treatment. We found that TNF-α significantly attenuated the increased levels of both apoA-IV mRNA (Figure 2A) and protein (Figure 2B) induced by LA, whereas IL-6 had no such effect on apoA-IV mRNA level (Figure 2A), but significantly attenuated apoA-IV protein level induced by 0.25 mM LA (Figure 2B). Furthermore, TNF-α and IL-6 themselves decreased apoA-IV protein levels (Figure 2B), but not apoA-IV mRNA levels (Figure 2A). These data indicate that both TNF-α and IL-6 inhibit the stimulatory effect of LA on apoA-IV production.Figure 2

Bottom Line: LA stimulated gene and protein expression of apoA-IV in a dose and time dependent manner.Pre-treatment with the cytokines for 72 h significantly inhibited the increased expression of apoA-IV gene and protein induced by LA.Our data indicate that the pro-inflammatory cytokines may be responsible for the reduced apoA-IV production in response to fat feeding.

View Article: PubMed Central - PubMed

Affiliation: National Local Joint Engineering Research Center of Biodiagnostics and Biotherapy, The Second Affiliated Hospital of Medical College, Xi'an Jiaotong University, Xi'an, 710004 China ; Department of Pathology and Laboratory Medicine, University of Cincinnati, Cincinnati, 45237-0507 USA.

ABSTRACT

Background: Apolipoprotein A-IV (apoA-IV) is a protein mainly synthesized by enterocytes in the intestine. Its gene expression is suppressed during fasting and stimulated during active fat absorption. Chronic feeding of a high-fat (HF) diet abolishes the differential expression between fasting and fat-feeding and therefore may contribute to diet-induced obesity since apoA-IV is a potent satiety factor. It is well established that the circulating pro-inflammatory cytokines TNF-α and IL-6 are increased by HF feeding.

Methods: To determine whether pro-inflammatory cytokines are involved in the diminished response of apoA-IV gene expression to fat-feeding, different concentrations of linoleic acid (LA), an important dietary fatty acid, was used to stimulate apoA-IV expression in human intestinal Caco2 cells. Cells were pre-treated with or without human recombinant TNF-α, IL-6 or their combination before the addition of LA. Real-time PCR and ELISA were used to detect and quantify RNA transcripts and proteins of apoA-IV and the cytokines.

Results: LA stimulated gene and protein expression of apoA-IV in a dose and time dependent manner. Pre-treatment with the cytokines for 72 h significantly inhibited the increased expression of apoA-IV gene and protein induced by LA. Furthermore, the cytokines, especially TNF-α, also positively up-regulate the cytokine themselves in Caco2 cells.

Conclusions: Our data indicate that the pro-inflammatory cytokines may be responsible for the reduced apoA-IV production in response to fat feeding. Because of apoA-IV's role in satiety, we propose the inhibitory effect of circulating pro-inflammatory cytokines on apoA-IV production contributes to diet-induced obesity.

No MeSH data available.


Related in: MedlinePlus