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Conjugated bile acid-activated S1P receptor 2 is a key regulator of sphingosine kinase 2 and hepatic gene expression - PubMed

. 2015 Apr;61(4):1216-26.

doi: 10.1002/hep.27592. Epub 2015 Mar 9.

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Conjugated bile acid-activated S1P receptor 2 is a key regulator of sphingosine kinase 2 and hepatic gene expression

Masayuki Nagahashi et al. Hepatology. 2015 Apr.

Abstract

Bile acids are important hormones during the feed/fast cycle, allowing the liver to coordinately regulate nutrient metabolism. How they accomplish this has not been fully elucidated. Conjugated bile acids activate both the ERK1/2 and AKT signaling pathways via sphingosine 1-phosphate receptor 2 (S1PR2) in rodent hepatocytes and in vivo. Here, we report that feeding mice a high-fat diet, infusion of taurocholate into the chronic bile fistula rat, or overexpression of the gene encoding S1PR2 in mouse hepatocytes significantly upregulated hepatic sphingosine kinase 2 (SphK2) but not SphK1. Key genes encoding nuclear receptors/enzymes involved in nutrient metabolism were significantly downregulated in livers of S1PR2(-/-) and SphK2(-/-) mice. In contrast, overexpression of the gene encoding S1PR2 in primary mouse hepatocytes differentially increased SphK2, but not SphK1, and mRNA levels of key genes involved in nutrient metabolism. Nuclear levels of sphingosine-1-phosphate, an endogenous inhibitor of histone deacetylases 1 and 2, as well as the acetylation of histones H3K9, H4K5, and H2BK12 were significantly decreased in hepatocytes prepared from S1PR2(-/-) and SphK2(-/-) mice.

Conclusion: Both S1PR2(-/-) and SphK2(-/-) mice rapidly developed fatty livers on a high-fat diet, suggesting the importance of conjugated bile acids, S1PR2, and SphK2 in regulating hepatic lipid metabolism.

© 2014 by the American Association for the Study of Liver Diseases.

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Conflict of interest statement

Competing Financial interest: The authors declare no competing financial interest.

Figures

Fig. 1
Fig. 1. Effect of SphK2 on hepatic histone acetylation levels and mRNA levels of key genes involved in hepatic lipid metabolism

(A) Nuclear extracts of primary hepatocytes isolated from Wild type (WT) and SphK2−/− mice were prepared as described in “Methods”. The protein levels of SphK2, H3K9ac, H4K5ac, H2BK12ac and total H3 were determined by Western blot analysis. Representative images are shown. (B) Total RNA was isolated from the livers of WT (solid bar) or SphK2−/− mice (checkered bar) (male, 20-week old). The mRNA levels of key genes involved in lipid metabolism were determined using real time RT-PCR and normalized using GAPDH. Abbs. SREBP-1c: sterol regulatory element-binding protein-1c; FAS: fatty acid synthase; LDLR: low-density lipoprotein receptor; CYP27A1: sterol 27-hydroxylase; CYP7A1: cholesterol 7 α-hydroxylase; BSEP: bile salt export pump; FXR: farnesoid X receptor; BECN1: autophagy-related gene (Atg) 6; PPARγ: peroxisome proliferator-activated receptor γ. *P<0.05, **P<0.01, ***P<0.01, statistical significance relative to WT mice, n=5. (C) Effect of SphK2 on association of acetylated H3K9 in SREBP1c and CYP7A1 chromatins. Mouse primary hepatocytes were isolated from wild type (WT) and SphK2−/− mice. ChIP assay and real-time PCR were used to quantify the DNA amount of SREBP1c and CYP7A1 associated with H3K9ac as described in “Methods”. Results were expressed as relative abundance of chromatin DNA associated with H3K9ac. Values are mean ± S.E. **P<0.01, statistical significance relative to WT, n=3. (D) The wild type mouse primary hepatocytes (solid bar) were treated with a chemical inhibitor of HDAC, SAHA (1 μM) for 8h (checkered bar). The total RNA was isolated. The mRNA levels of key genes involved in lipid metabolism were determined using real time PCR and normalized using β-actin. CPT-1α: Carnitine palmitoyltransferase I α; PCG1α: Peroxisome proliferator-activated receptor gamma coactivator 1-alpha; ACC1: Acetyl-CoA carboxylase 1.

Fig. 2
Fig. 2. Effect of high fat diet and cholic acid on SphK2 expression and enzymatic activity in liver

Wild type (WT) and SphK2−/− (KO) mice (male, 20-week old) were fed normal chow diet (ND; solid bar), high fat diet (HFD; checkered bar), or HFD containing 1% cholic acid (HFD+CA; shaded bar) for two weeks. (A) The mRNA levels of SphK2 in the liver of WT mice were determined by real time RT-PCR and normalized using GAPDH as internal control. (B) The protein expression levels of SphK2 in the liver of WT and SphK2−/− mice were determined by Western Blot analysis and normalized using β-tubulin as loading control. (C) The enzyme activities of SphK2 were measured using [γ32P]-ATP. *P<0.05, **P<0.01, statistical significance relative to control group.

Fig. 3
Fig. 3. Induction of SphK2 by TCA in the chronic bile fistula rat

Bile fistulas were placed in rats and the bile was drained for 48 hrs. TCA was infused intraduodenally at a rate of 1.05 ml/h/100 g rat and a concentration of 36 μmol/h/100 g rat for 4h (checkered bar). Animals were harvested and the liver pieces from each animal were isolated and snap-frozen. The protein levels of SphK1 and SphK2 were detected by Western blot analysis using antibodies against SphK1 and SphK2 as described in “Methods”. (A) Representative images of immunoblots for SphK1, SphK2 and actin are shown. Relative densities of SphK1 and SphK2 were determined by scanning laser density spectrometry and actin was used as a loading control. (B and C) The specific enzyme activities of SphK1 and SphK2 were determined using [γ32P]-ATP and sphingosine as described in “Methods”. *p<0.05, statistical significance relative to control group, n=5.

Fig. 4
Fig. 4. Effect of S1PR2 on SphK2 expression and enzymatic activity

(A) Total RNA was isolated from the liver of Wild type (WT) and S1PR2−/− mice. The mRNA levels of SphK2 were determined using real-time RT-PCR. (B) The total liver protein lysates of WT and S1PR2−/− mice were prepared and protein levels of SphK1 and SphK2 were determined by Western blot analysis. Representative images are shown. (C) The relative densities of SphK1 (solid bar) and SphK2 (checkered bar) were determined using β-tubulin as a loading control. (D) Primary mouse hepatocytes were isolated from Wild type (WT) and S1PR2−/− mice. The protein levels of SphK1 and SphK2 were determined by Western Blot analysis and the representative images are shown. β-tubulin was used as a loading control. (E and F) Primary mouse hepatocytes isolated from WT (closed circle) and S1PR2−/− (closed diamond) mice were treated with TCA (100 μM) for 0–30 min). The enzyme activities of SphK1 and SphK2 were measured using [γ32P]-ATP and sphingosine.

Fig. 5
Fig. 5. Effect of S1PR2 on hepatic histone acetylation levels and mRNA levels of key genes involved in hepatic lipid metabolism

(A) Nuclear extracts of primary hepatocytes isolated from Wild type (WT) and S1PR2−/− mice were prepared as described in “Methods”. The protein levels of SphK2, H3K9ac, H4K5ac, H2BK12ac and total H3 were determined by Western blot analysis. Representative images are shown. (B) Total RNA was isolated from the livers of WT (solid bar) or S1PR2−/− (checkered bar) mice (male, 20-week old). The mRNA levels of key genes involved in lipid metabolism were determined using real time RT-PCR and normalized using β-actin. Abbs: SREBP-1c: sterol regulatory element-binding protein-1c; FAS: fatty acid synthase; LDLR: low-density lipoprotein receptor; CYP27A1: sterol 27-hydroxylase; CYP7A1: cholesterol 7 α-hydroxylase; BSEP: bile salt export pump; FXR: farnesoid X receptor. BECN1: autophagy-related gene (Atg) 6; PPARγ: peroxisome proliferator-activated receptor γ. *P<0.05, **P<0.01, ***P<0.01, statistical significance relative to WT mice, n=5–8. (C and D) The S1P and DHS1P levels in the cytosol and nucleus of mouse primary hepatocytes isolated from WT and S1PR2−/− mice were measured using mass spectrometry as described in “Methods”. *P<0.05, statistical significance relative to WT.

Fig. 6
Fig. 6. Effect of overexpression of the gene encoding S1PR2 on gene regulation in mouse primary hepatocytes

The mouse primary hepatocytes isolated from wild type (WT) or S1PR2−/− mice were transfected with a control vector or pcDNA3-mS1PR2 as described in “Methods”. The total cellular RNA was isolated after 48h. The mRNA levels of specific target genes were determined using real-time RT-PCR and normalized using GAPDH as an internal control. (A) The mRNA levels of S1PR2; (B) The mRNA levels of SphK1 and SphK2; (C) The mRNA levels of key genes involved in lipid metabolism. *P<0.05, **P<0.01, ***P<0.001, statistical significance relative to control vector transfected cells. (D) Effect of overexpression of the gene encoding S1PR2 on gene regulation in SphK2−/− mouse primary hepatocytes. The mouse primary hepatocytes isolated from SphK2−/− mice were transfected with a control vector or pcDNA3-mS1PR2 as described above. The total cellular RNA was isolated after 48h. The mRNA levels of specific target genes were determined using real-time RT-PCR and normalized using GAPDH as an internal control. The mRNA levels of key genes involved in lipid metabolism. *P<0.05, **P<0.01, ***P<0.001, statistical significance relative to control vector transfected cells.

Fig. 7
Fig. 7. Effect of HFD on hepatic lipid accumulation in S1PR2−/− SphK2−/− mice

Wild type (WT), S1PR2−/− and SphK2−/− mice (male, 20-week old) were fed HFD for two weeks. The liver sections were stained using Oil Red O or HE. The images were taken with an Olympus microscope equipped with image recorder using a 400x lens. Representative images of livers, HE staining and Oil Red O staining are shown. (A) S1PR2−/− mice; (B) SphK2−/− mice.

Fig. 8
Fig. 8. Model of CBA regulation of hepatic genes encoding enzymes involved in nutrient metabolism

CBA returning from the intestines following a meal activates S1PR2. Activation of this GPCR then activates nuclear SphK2 via cell signaling pathways increasing the levels of S1P in the nucleus. Nuclear S1P inhibits specific histone deacetylases (HDACs) causing an increase in acetylation of histones and up-regulation of genes encoding nuclear receptors and enzymes involved in nutrient metabolism. This CBA-activated nutrient signaling pathway is hypothesized to allow the liver to be more efficient in metabolizing a bolus of incoming nutrients allowing the liver to maintain “nutrient homeostasis”.

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