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Regulation of IGFBP-2 expression during fasting.

Kang HS, Kim MY, Kim SJ, Lee JH, Kim YD, Seo YK, Bae JH, Oh GT, Song DK, Ahn YH, Im SS - Biochem. J. (2015)

Bottom Line: Wy14643, a selective PPARα agonist, significantly induced Igfbp-2 gene expression in primary cultured hepatocytes.To explore the role of PPARα in IGF-1 signalling, we treated primary cultured hepatocytes with Wy14643 and observed a decrease in the number of IGF-1 receptors (IGF-1Rs) and in Akt phosphorylation.These results suggest that PPARα controls IGF-1 signalling through the up-regulation of hepatic Igfbp-2 transcription during fasting and Wy14643 treatment.

View Article: PubMed Central - PubMed

Affiliation: *Department of Physiology, Keimyung University School of Medicine, Daegu 704-701, Republic of Korea.

ABSTRACT
Insulin-like growth factor (IGF)-binding protein-2 (IGFBP-2), one of the most abundant circulating IGFBPs, is known to attenuate the biological action of IGF-1. Although the effect of IGFBP-2 in preventing metabolic disorders is well known, its regulatory mechanism remains unclear. In the present study, we demonstrated the transcriptional regulation of the Igfbp-2 gene by peroxisome-proliferator-activated receptor (PPAR) α in the liver. During fasting, both Igfbp-2 and PPARα expression levels were increased. Wy14643, a selective PPARα agonist, significantly induced Igfbp-2 gene expression in primary cultured hepatocytes. However, Igfbp-2 gene expression in Pparα mice was not affected by fasting or Wy14643. In addition, through transient transfection and chromatin immunoprecipitation assay in fasted livers, we determined that PPARα bound to the putative PPAR-responsive element between -511 bp and -499 bp on the Igfbp-2 gene promoter, indicating that the Igfbp-2 gene transcription is activated directly by PPARα. To explore the role of PPARα in IGF-1 signalling, we treated primary cultured hepatocytes with Wy14643 and observed a decrease in the number of IGF-1 receptors (IGF-1Rs) and in Akt phosphorylation. No inhibition was observed in the hepatocytes isolated from Pparα mice. These results suggest that PPARα controls IGF-1 signalling through the up-regulation of hepatic Igfbp-2 transcription during fasting and Wy14643 treatment.

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PPARα is involved in the induction of IGFBP-2 following the fasting state and Wy14643 treatment(A) Mouse primary hepatocytes were treated with Wy14643 (Wy) for 6 h at the indicated concentrations. Total RNA was isolated and analysed using qPCR with the observed primers. (B) Mouse primary hepatocytes from WT and Pparα  mice were treated with or without Wy14643 for 6 h. The level of Pparα mRNA was measured by qPCR analysis. (C) The mRNA level of Igfbp-2 in liver from WT and Pparα  mice fed and fasted for 24 h was analysed by qPCR. (D) Protein level of IGFBP-2 in livers from WT and Pparα  mice fed and fasted for 24 h was analysed by Western blot analysis. (E) Secretion level of IGFBP-2 in vivo and in vitro. Mice were fed or fasted for 24 h and serum was collected from WT and Pparα  mice. Secretion levels of IGFBP-2 in feeding or fasting conditions were measured by ELISA. (F) WT and Pparα  mice were treated with or without Wy14643 for 6 h. Whole cell extracts were isolated from primary hepatocytes of the observed conditions and assessed by Western blot analysis with the indicated antibody. (G–I) Primary hepatocytes from WT and Pparα  mice were treated with or without Wy14643 for 6 h. The level of Pparγ1, PPARγ2 and PPARδ mRNA was measured by qPCR analysis. *P<0.05 and **P<0.01 compared with untreated control or fed WT mice.
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Figure 2: PPARα is involved in the induction of IGFBP-2 following the fasting state and Wy14643 treatment(A) Mouse primary hepatocytes were treated with Wy14643 (Wy) for 6 h at the indicated concentrations. Total RNA was isolated and analysed using qPCR with the observed primers. (B) Mouse primary hepatocytes from WT and Pparα mice were treated with or without Wy14643 for 6 h. The level of Pparα mRNA was measured by qPCR analysis. (C) The mRNA level of Igfbp-2 in liver from WT and Pparα mice fed and fasted for 24 h was analysed by qPCR. (D) Protein level of IGFBP-2 in livers from WT and Pparα mice fed and fasted for 24 h was analysed by Western blot analysis. (E) Secretion level of IGFBP-2 in vivo and in vitro. Mice were fed or fasted for 24 h and serum was collected from WT and Pparα mice. Secretion levels of IGFBP-2 in feeding or fasting conditions were measured by ELISA. (F) WT and Pparα mice were treated with or without Wy14643 for 6 h. Whole cell extracts were isolated from primary hepatocytes of the observed conditions and assessed by Western blot analysis with the indicated antibody. (G–I) Primary hepatocytes from WT and Pparα mice were treated with or without Wy14643 for 6 h. The level of Pparγ1, PPARγ2 and PPARδ mRNA was measured by qPCR analysis. *P<0.05 and **P<0.01 compared with untreated control or fed WT mice.

Mentions: Next, we examined whether PPARα plays a role in regulating Igfbp-2 gene expression in primary cultured hepatocytes. Wy14643 treatment in hepatocytes resulted in a significant increase in the mRNA levels of Igfbp-2 and Pparα in a dose-dependent manner, but did not affect Igfbp-1 (Figure 2A). In addition, Wy14643 treatment increased Pparα mRNA levels in the hepatocytes of WT mice but not in Pparα mice (Figure 2B). Moreover, Igfbp-2 mRNA expression increased in fasting WT mice compared with fed or refed mice, in a fashion similar to that shown in Figure 1. However, this phenomenon was not observed in Pparα mice (Figures 2C and 2D). Next, we measured the level of secreted IGFBP-2 in the serum of WT and PPARα mice subjected to feeding and fasting. The level of IGFBP-2 in the serum was increased in the fasted WT mice (Figure 2E) but not in the Pparα mice. This indicates that IGFBP-2 secretion as well as mRNA expression of Igfbp-2 is also increased by PPARα. In accordance with mRNA level, the protein level of IGFBP-2 increased prominently in Wy14643-treated WT mice. Again, this increase in protein level was not observed in Wy14643-treated Pparα mice (Figure 2F). Other PPAR isoforms were not affected by Wy14643 treatment in the primary hepatocytes (Figures 2G–2I). Collectively, these results indicate that PPARα is a key mediator for up-regulating IGFBP-2 expression in primary cultured hepatocytes.


Regulation of IGFBP-2 expression during fasting.

Kang HS, Kim MY, Kim SJ, Lee JH, Kim YD, Seo YK, Bae JH, Oh GT, Song DK, Ahn YH, Im SS - Biochem. J. (2015)

PPARα is involved in the induction of IGFBP-2 following the fasting state and Wy14643 treatment(A) Mouse primary hepatocytes were treated with Wy14643 (Wy) for 6 h at the indicated concentrations. Total RNA was isolated and analysed using qPCR with the observed primers. (B) Mouse primary hepatocytes from WT and Pparα  mice were treated with or without Wy14643 for 6 h. The level of Pparα mRNA was measured by qPCR analysis. (C) The mRNA level of Igfbp-2 in liver from WT and Pparα  mice fed and fasted for 24 h was analysed by qPCR. (D) Protein level of IGFBP-2 in livers from WT and Pparα  mice fed and fasted for 24 h was analysed by Western blot analysis. (E) Secretion level of IGFBP-2 in vivo and in vitro. Mice were fed or fasted for 24 h and serum was collected from WT and Pparα  mice. Secretion levels of IGFBP-2 in feeding or fasting conditions were measured by ELISA. (F) WT and Pparα  mice were treated with or without Wy14643 for 6 h. Whole cell extracts were isolated from primary hepatocytes of the observed conditions and assessed by Western blot analysis with the indicated antibody. (G–I) Primary hepatocytes from WT and Pparα  mice were treated with or without Wy14643 for 6 h. The level of Pparγ1, PPARγ2 and PPARδ mRNA was measured by qPCR analysis. *P<0.05 and **P<0.01 compared with untreated control or fed WT mice.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 2: PPARα is involved in the induction of IGFBP-2 following the fasting state and Wy14643 treatment(A) Mouse primary hepatocytes were treated with Wy14643 (Wy) for 6 h at the indicated concentrations. Total RNA was isolated and analysed using qPCR with the observed primers. (B) Mouse primary hepatocytes from WT and Pparα mice were treated with or without Wy14643 for 6 h. The level of Pparα mRNA was measured by qPCR analysis. (C) The mRNA level of Igfbp-2 in liver from WT and Pparα mice fed and fasted for 24 h was analysed by qPCR. (D) Protein level of IGFBP-2 in livers from WT and Pparα mice fed and fasted for 24 h was analysed by Western blot analysis. (E) Secretion level of IGFBP-2 in vivo and in vitro. Mice were fed or fasted for 24 h and serum was collected from WT and Pparα mice. Secretion levels of IGFBP-2 in feeding or fasting conditions were measured by ELISA. (F) WT and Pparα mice were treated with or without Wy14643 for 6 h. Whole cell extracts were isolated from primary hepatocytes of the observed conditions and assessed by Western blot analysis with the indicated antibody. (G–I) Primary hepatocytes from WT and Pparα mice were treated with or without Wy14643 for 6 h. The level of Pparγ1, PPARγ2 and PPARδ mRNA was measured by qPCR analysis. *P<0.05 and **P<0.01 compared with untreated control or fed WT mice.
Mentions: Next, we examined whether PPARα plays a role in regulating Igfbp-2 gene expression in primary cultured hepatocytes. Wy14643 treatment in hepatocytes resulted in a significant increase in the mRNA levels of Igfbp-2 and Pparα in a dose-dependent manner, but did not affect Igfbp-1 (Figure 2A). In addition, Wy14643 treatment increased Pparα mRNA levels in the hepatocytes of WT mice but not in Pparα mice (Figure 2B). Moreover, Igfbp-2 mRNA expression increased in fasting WT mice compared with fed or refed mice, in a fashion similar to that shown in Figure 1. However, this phenomenon was not observed in Pparα mice (Figures 2C and 2D). Next, we measured the level of secreted IGFBP-2 in the serum of WT and PPARα mice subjected to feeding and fasting. The level of IGFBP-2 in the serum was increased in the fasted WT mice (Figure 2E) but not in the Pparα mice. This indicates that IGFBP-2 secretion as well as mRNA expression of Igfbp-2 is also increased by PPARα. In accordance with mRNA level, the protein level of IGFBP-2 increased prominently in Wy14643-treated WT mice. Again, this increase in protein level was not observed in Wy14643-treated Pparα mice (Figure 2F). Other PPAR isoforms were not affected by Wy14643 treatment in the primary hepatocytes (Figures 2G–2I). Collectively, these results indicate that PPARα is a key mediator for up-regulating IGFBP-2 expression in primary cultured hepatocytes.

Bottom Line: Wy14643, a selective PPARα agonist, significantly induced Igfbp-2 gene expression in primary cultured hepatocytes.To explore the role of PPARα in IGF-1 signalling, we treated primary cultured hepatocytes with Wy14643 and observed a decrease in the number of IGF-1 receptors (IGF-1Rs) and in Akt phosphorylation.These results suggest that PPARα controls IGF-1 signalling through the up-regulation of hepatic Igfbp-2 transcription during fasting and Wy14643 treatment.

View Article: PubMed Central - PubMed

Affiliation: *Department of Physiology, Keimyung University School of Medicine, Daegu 704-701, Republic of Korea.

ABSTRACT
Insulin-like growth factor (IGF)-binding protein-2 (IGFBP-2), one of the most abundant circulating IGFBPs, is known to attenuate the biological action of IGF-1. Although the effect of IGFBP-2 in preventing metabolic disorders is well known, its regulatory mechanism remains unclear. In the present study, we demonstrated the transcriptional regulation of the Igfbp-2 gene by peroxisome-proliferator-activated receptor (PPAR) α in the liver. During fasting, both Igfbp-2 and PPARα expression levels were increased. Wy14643, a selective PPARα agonist, significantly induced Igfbp-2 gene expression in primary cultured hepatocytes. However, Igfbp-2 gene expression in Pparα mice was not affected by fasting or Wy14643. In addition, through transient transfection and chromatin immunoprecipitation assay in fasted livers, we determined that PPARα bound to the putative PPAR-responsive element between -511 bp and -499 bp on the Igfbp-2 gene promoter, indicating that the Igfbp-2 gene transcription is activated directly by PPARα. To explore the role of PPARα in IGF-1 signalling, we treated primary cultured hepatocytes with Wy14643 and observed a decrease in the number of IGF-1 receptors (IGF-1Rs) and in Akt phosphorylation. No inhibition was observed in the hepatocytes isolated from Pparα mice. These results suggest that PPARα controls IGF-1 signalling through the up-regulation of hepatic Igfbp-2 transcription during fasting and Wy14643 treatment.

Show MeSH
Related in: MedlinePlus