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Apolipoprotein D Transgenic Mice Develop Hepatic Steatosis through Activation of PPARγ and Fatty Acid Uptake.

Labrie M, Lalonde S, Najyb O, Thiery M, Daneault C, Des Rosiers C, Rassart E, Mounier C - PLoS ONE (2015)

Bottom Line: Elevated expression of AMPK in the liver of Tg leads to phosphorylation of acetyl CoA carboxylase, indicating a decreased activity of the enzyme.In addition, expression of carnitine palmitoyl transferase 1, the rate-limiting enzyme of beta-oxidation, is slightly upregulated.Finally, we show that overexpressing H-apoD in HepG2 cells in presence of arachidonic acid (AA), the main apoD ligand, increases the transcriptional activity of PPARγ.

View Article: PubMed Central - PubMed

Affiliation: Centre de recherche BioMed, Département des Sciences Biologiques, Université du Québec, Montréal, Québec, H3C 3P8, Canada.

ABSTRACT
Transgenic mice (Tg) overexpressing human apolipoprotein D (H-apoD) in the brain are resistant to neurodegeneration. Despite the use of a neuron-specific promoter to generate the Tg mice, they expressed significant levels of H-apoD in both plasma and liver and they slowly develop hepatic steatosis and insulin resistance. We show here that hepatic PPARγ expression in Tg mice is increased by 2-fold compared to wild type (WT) mice. Consequently, PPARγ target genes Plin2 and Cide A/C are overexpressed, leading to increased lipid droplets formation. Expression of the fatty acid transporter CD36, another PPARgamma target, is also increased in Tg mice associated with elevated fatty acid uptake as measured in primary hepatocytes. Elevated expression of AMPK in the liver of Tg leads to phosphorylation of acetyl CoA carboxylase, indicating a decreased activity of the enzyme. Fatty acid synthase expression is also induced but the hepatic lipogenesis measured in vivo is not significantly different between WT and Tg mice. In addition, expression of carnitine palmitoyl transferase 1, the rate-limiting enzyme of beta-oxidation, is slightly upregulated. Finally, we show that overexpressing H-apoD in HepG2 cells in presence of arachidonic acid (AA), the main apoD ligand, increases the transcriptional activity of PPARγ. Supporting the role of apoD in AA transport, we observed enrichment in hepatic AA and a decrease in plasmatic AA concentration. Taken together, our results demonstrate that the hepatic steatosis observed in apoD Tg mice is a consequence of increased PPARγ transcriptional activity by AA leading to increased fatty acid uptake by the liver.

No MeSH data available.


Related in: MedlinePlus

PPARγ transcriptional activity in presence of AA and/or apoD.A- HepG2 cells were either non transfected (NT) or transfected with a myc-Tag apoD-cDNA or empty vector (EV) construct and incubated with BSA or arachidonic acid (AA). The level of H-apoD expression was evaluated by Western blot using a specific H-apoD antibody. B- HepG2 cells were transfected with UAS-Luc, GAL4-PPARγ, β-galactosidase and with either an empty vector or a myc-Tag apoD-cDNA construct. After transfection, cells were treated with 7 μM AA for 4h. Luciferase activity represents data normalized by β-galactosidase activity. The data represent the mean ± SD (n = 3). *P<0.05 and **P<0.01 vs the non-stimulated control without apoD. The gel presented below showed the expression of apoD in transfected cells using a myc antibody.
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pone.0130230.g006: PPARγ transcriptional activity in presence of AA and/or apoD.A- HepG2 cells were either non transfected (NT) or transfected with a myc-Tag apoD-cDNA or empty vector (EV) construct and incubated with BSA or arachidonic acid (AA). The level of H-apoD expression was evaluated by Western blot using a specific H-apoD antibody. B- HepG2 cells were transfected with UAS-Luc, GAL4-PPARγ, β-galactosidase and with either an empty vector or a myc-Tag apoD-cDNA construct. After transfection, cells were treated with 7 μM AA for 4h. Luciferase activity represents data normalized by β-galactosidase activity. The data represent the mean ± SD (n = 3). *P<0.05 and **P<0.01 vs the non-stimulated control without apoD. The gel presented below showed the expression of apoD in transfected cells using a myc antibody.

Mentions: To understand the link between apoD and PPARγ over-expression and activation, we evaluated the potential role of apoD as an AA transporter, one of the main ligands of PPARγ. We used the human hepatocarcinoma HepG2 cell line as a well-characterized model for the study of hepatic lipid metabolism. Cells were transfected with a construct containing the cDNA of human apoD. No apoD was detected in cells either untransfected or transfected with an empty vector. Transfection with the H-apoD cDNA showed a strong expression of the protein (Fig 6A). We next co-transfected the H-apoD cDNA construct with a construct containing five peroxisomes proliferator-activator receptor elements (PPRE) linked to a luciferase reporter gene. Thereafter, cells were incubated with 7 μM of AA in presence or absence of apoD. At this concentration, AA does not induce any cellular toxicity [53] but a slight decrease in apoD expression (Fig 6A). Over-expression of apoD increased PPARγ transcriptional activity (3.7 fold, Fig 6B). Addition of AA alone increased PPARγ transcriptional activity to a similar extent (3.9-fold). Very interestingly, a combination of AA and apoD over-expression showed a very strong synergistic transactivation effect on PPARγ transcriptional activity (approximately 9-fold) (Fig 6B).


Apolipoprotein D Transgenic Mice Develop Hepatic Steatosis through Activation of PPARγ and Fatty Acid Uptake.

Labrie M, Lalonde S, Najyb O, Thiery M, Daneault C, Des Rosiers C, Rassart E, Mounier C - PLoS ONE (2015)

PPARγ transcriptional activity in presence of AA and/or apoD.A- HepG2 cells were either non transfected (NT) or transfected with a myc-Tag apoD-cDNA or empty vector (EV) construct and incubated with BSA or arachidonic acid (AA). The level of H-apoD expression was evaluated by Western blot using a specific H-apoD antibody. B- HepG2 cells were transfected with UAS-Luc, GAL4-PPARγ, β-galactosidase and with either an empty vector or a myc-Tag apoD-cDNA construct. After transfection, cells were treated with 7 μM AA for 4h. Luciferase activity represents data normalized by β-galactosidase activity. The data represent the mean ± SD (n = 3). *P<0.05 and **P<0.01 vs the non-stimulated control without apoD. The gel presented below showed the expression of apoD in transfected cells using a myc antibody.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4470830&req=5

pone.0130230.g006: PPARγ transcriptional activity in presence of AA and/or apoD.A- HepG2 cells were either non transfected (NT) or transfected with a myc-Tag apoD-cDNA or empty vector (EV) construct and incubated with BSA or arachidonic acid (AA). The level of H-apoD expression was evaluated by Western blot using a specific H-apoD antibody. B- HepG2 cells were transfected with UAS-Luc, GAL4-PPARγ, β-galactosidase and with either an empty vector or a myc-Tag apoD-cDNA construct. After transfection, cells were treated with 7 μM AA for 4h. Luciferase activity represents data normalized by β-galactosidase activity. The data represent the mean ± SD (n = 3). *P<0.05 and **P<0.01 vs the non-stimulated control without apoD. The gel presented below showed the expression of apoD in transfected cells using a myc antibody.
Mentions: To understand the link between apoD and PPARγ over-expression and activation, we evaluated the potential role of apoD as an AA transporter, one of the main ligands of PPARγ. We used the human hepatocarcinoma HepG2 cell line as a well-characterized model for the study of hepatic lipid metabolism. Cells were transfected with a construct containing the cDNA of human apoD. No apoD was detected in cells either untransfected or transfected with an empty vector. Transfection with the H-apoD cDNA showed a strong expression of the protein (Fig 6A). We next co-transfected the H-apoD cDNA construct with a construct containing five peroxisomes proliferator-activator receptor elements (PPRE) linked to a luciferase reporter gene. Thereafter, cells were incubated with 7 μM of AA in presence or absence of apoD. At this concentration, AA does not induce any cellular toxicity [53] but a slight decrease in apoD expression (Fig 6A). Over-expression of apoD increased PPARγ transcriptional activity (3.7 fold, Fig 6B). Addition of AA alone increased PPARγ transcriptional activity to a similar extent (3.9-fold). Very interestingly, a combination of AA and apoD over-expression showed a very strong synergistic transactivation effect on PPARγ transcriptional activity (approximately 9-fold) (Fig 6B).

Bottom Line: Elevated expression of AMPK in the liver of Tg leads to phosphorylation of acetyl CoA carboxylase, indicating a decreased activity of the enzyme.In addition, expression of carnitine palmitoyl transferase 1, the rate-limiting enzyme of beta-oxidation, is slightly upregulated.Finally, we show that overexpressing H-apoD in HepG2 cells in presence of arachidonic acid (AA), the main apoD ligand, increases the transcriptional activity of PPARγ.

View Article: PubMed Central - PubMed

Affiliation: Centre de recherche BioMed, Département des Sciences Biologiques, Université du Québec, Montréal, Québec, H3C 3P8, Canada.

ABSTRACT
Transgenic mice (Tg) overexpressing human apolipoprotein D (H-apoD) in the brain are resistant to neurodegeneration. Despite the use of a neuron-specific promoter to generate the Tg mice, they expressed significant levels of H-apoD in both plasma and liver and they slowly develop hepatic steatosis and insulin resistance. We show here that hepatic PPARγ expression in Tg mice is increased by 2-fold compared to wild type (WT) mice. Consequently, PPARγ target genes Plin2 and Cide A/C are overexpressed, leading to increased lipid droplets formation. Expression of the fatty acid transporter CD36, another PPARgamma target, is also increased in Tg mice associated with elevated fatty acid uptake as measured in primary hepatocytes. Elevated expression of AMPK in the liver of Tg leads to phosphorylation of acetyl CoA carboxylase, indicating a decreased activity of the enzyme. Fatty acid synthase expression is also induced but the hepatic lipogenesis measured in vivo is not significantly different between WT and Tg mice. In addition, expression of carnitine palmitoyl transferase 1, the rate-limiting enzyme of beta-oxidation, is slightly upregulated. Finally, we show that overexpressing H-apoD in HepG2 cells in presence of arachidonic acid (AA), the main apoD ligand, increases the transcriptional activity of PPARγ. Supporting the role of apoD in AA transport, we observed enrichment in hepatic AA and a decrease in plasmatic AA concentration. Taken together, our results demonstrate that the hepatic steatosis observed in apoD Tg mice is a consequence of increased PPARγ transcriptional activity by AA leading to increased fatty acid uptake by the liver.

No MeSH data available.


Related in: MedlinePlus