Role of PPARα and HNF4α in Stress-Mediated Alterations in Lipid Homeostasis

<div><p>Stress is a risk factor for several cardiovascular pathologies. PPARα holds a fundamental role in control of lipid homeostasis by directly regulating genes involved in fatty acid transport and oxidation. Importantly, PPARα agonists are effective in raising HDL-cholesterol and lowering triglycerides, properties that reduce the risk for cardiovascular diseases. This study investigated the role of stress and adrenergic receptor (AR)-related pathways in PPARα and HNF4α regulation and signaling in mice following repeated restraint stress or treatment with AR-antagonists administered prior to stress to block AR-linked pathways. Repeated restraint stress up-regulated <i>Pparα</i> and its target genes in the liver, including <i>Acox</i>, <i>Acot1</i>, <i>Acot4</i>, <i>Cyp4a10</i>, <i>Cyp4a14 and Lipin2</i>, an effect that was highly correlated with <i>Hnf4α</i>. <i>In vitro</i> studies using primary hepatocyte cultures treated with epinephrine or AR-agonists confirmed that hepatic AR/cAMP/PKA/CREB- and JNK-linked pathways are involved in PPARα and HNF4α regulation. Notably, restraint stress, independent of PPARα, suppressed plasma triglyceride levels. This stress-induced effect could be attributed in part to hormone sensitive lipase activation in the white adipose tissue, which was not prevented by the increased levels of perilipin. Overall, this study identifies a mechanistic basis for the modification of lipid homeostasis following stress and potentially indicates novel roles for PPARα and HNF4α in stress-induced lipid metabolism.</p></div

Stress-induced effect on hepatic TG homeostasis.

<p>(A) Effect of restraint stress on genes involved in TG synthesis and lipolysis in the liver. (B) Effect of restraint stress on genes involved in TG metabolism and clearance in the liver. Comparisons were between controls and stress-exposed mice (alone or simultaneously treated with the AR-antagonists, prazosin, propranolol or atipamezole; black bars). DGAT1: Diacyl glycerol acyltransferase 1 (acyl coenzyme A (CoA)), DGAT2: Diacyl glycerol acyltransferase 2, LPL: lipoprotein lipase, HSL: hormone sensitive lipase, ATGL/PNPLA2: adipose triglyceride lipase/patatin-like phospholipase domain containing 2, orphan nuclear receptor NR4A, AADAC: arylacetamide deacetylase, CD36: cluster of differentiation 36 or fatty acid transporter, CES3/TGH: carboxylesterase 3, MTTP: microsomal triglyceride transfer, PLIN5: perilipin 5. C: Control, N.Saline: normal saline, Prazosin (alpha₁-AR antagonist), Atipamezole (alpha₂-AR antagonist), Propranolol (beta-AR antagonist). Values are expressed as mean ± SE, n:5–6 per treatment group. *P<0.005, **P<0.01, ***P<0.001.</p

Stress-induced effect on <i>PPARα</i> and target gene expression.

<p>A. Following exposure of wild-type mice to restraint stress, <i>Ppara</i> mRNA levels were examined by qPCR analysis in their liver and PPARα protein was measured in the nuclear fraction by Western Blot analysis. Histone H1 served as a loading control. B. <i>Acox, Cyp4a10, Cyp4a14, Lipin2, Acot1</i>, <i>Acot4</i>, <i>Lipin1</i> and <i>RXRα</i> mRNA levels were also analyzed by qPCR in wild-type mice. Values were quantified using the comparative CT methods normalized to β-actin and are expressed as mean ± SE (n = 8–10). Comparisons were between controls and stress-exposed mice (alone or previously treated with the AR-antagonists, prazosin, propranolol or atipamezole). Group differences were calculated by one-way ANOVA, followed by Bonferonni's test. * P<0.025, **P<0.01, ***P<0.001.</p

Stress-induced effect on PI3K/AKT, cAMP/PKA/CREB and GH/STAT5b signaling pathways.

<p>The evaluation of the stress effect on theses pathways was conducted in nuclear and cytosolic proteins with Western blot. Histone H3 served as a loading control for nuclear proteins and β-actin for cytosolic proteins. AR: adrenergic receptor; AR-antagonists given prior to stress inhibited α₁-, α₂- and beta-AR signaling; prazosin (α₁-AR ainhibitor), atipamezole (α₂-AR inhibitor), propranolol (β-AR inhibitor).</p

Stress-induced effect on <i>Hnf4a</i> expression.

<p>A. Following restraint stress alone or coupled with AR-antagonists hepatic <i>Hnf4a</i> mRNA levels were analysed in wild-type mice by qPCR. HNF4α protein was measured in liver nuclear fractions by Western blot analysis. Histone H1 served as a loading control. B. <i>Cyp8b1</i> and <i>Baat</i> mRNA levels were analyzed in the livers of wild-type mice by qPCR following treatment with either restraint stress alone or coupled with AR-antagonists (dark bars). C. <i>Hnf4a</i> mRNA levels were determined by qPCR following treatment of primary hepatocyte cultures with either corticosterone (CORT), epinephrine (EPIN) or AR-agonists for 24 hours. Primary hepatocytes were also treated with AR-agonists in combination with the JNK inhibitor, SP600125 (SP), or the PKA inhibitors, H89 or sodium orthovanadate (NaOV). Values were normalized to β-actin and are expressed as mean ± SE (n = 8–10). Comparisons were between controls and stress- or drug-treated mice. AR: adrenergic receptor, C: control, N. Saline: normal saline, All inhibitors: mice were treated with all AR-antagonists prior to stress, Prazosin (alpha₁-AR antagonist), Atipamezole (alpha₂-AR antagonist), Propranolol (beta-AR antagonist), PH: Phenylephrine (alpha₁-AR agonist), ISOP: Isoprenaline (beta-AR agonist). Group differences were calculated by one-way ANOVA, followed by Bonferonni's test. *P<0.025, **P<0.01, ***P<0.001.</p

Role of PPARα in the stress-induced changes in serum lipid markers.

<p>Triglycerides (TG), free fatty acids (FFA) and total cholesterol (T-cholesterol). Alanine aminotransferase (ALT), aspartate aminotransferase (AST), corticosterone (CORT), epinephrine (EPIN), norepinephrine (NE), (WT, n = 20; <i>Ppara</i>-null, n = 12).</p>*<p>P<0.05,</p>**<p>P<0.01.</p

<i>In vitro</i> evaluation of the role of glucocorticoids and hepatic adrenergic receptor-linked pathways in <i>PPARα</i> and target gene expression.

<p>α. Following treatment of primary hepatocyte cultures with adrenergic receptor agonists for 24 hours, <i>Acox, Cyp4a10, Cyp4a14, Acot1, Acot4</i>, <i>Lipin1</i> and <i>Lipin2</i> mRNA levels were analyzed by qPCR. B. <i>Ppara</i> mRNA levels were also determined by qPCR in primary hepatocytes treated with either corticosterone, epinephrine or AR-agonists for 24 hours. Primary hepatocytes were also treated with AR-agonists in combination with the JNK inhibitor, SP600125 (SP), or the PKA inhibitors, H89 or sodium orthovanadate (NaOV). Values were quantified using the comparative CT methods normalized to <i>β</i>-actin and are expressed as mean ± SE (n = 3–4). Experiments were repeated three times. Comparisons were between control (DMSO) and drug-treated hepatocytes. C: control, CORT: corticosterone, PH: phenylephrine (alpha₁-AR agonist), ISOP: isoprenaline (beta-AR agonist), EPIN: epinephrine (alpha- and beta-AR agonist), cAMP: 8-Br-cAMP. Group differences were calculated by one-way ANOVA, followed by Bonferonni's test. * P<0.025, **P<0.01, ***P<0.001.</p

Stress-induced effect on <i>Acadm</i> expression.

<p>Effect of restraint stress and AR-related pathways on <i>Acadm</i> mRNA expression in the liver and in white adipose tissue (A), on Pcsk9 and Ldlr mRNA expression in the liver (B). Values were normalized to β-actin and are expressed as mean ± SE (n = 8–10). Comparisons were between controls and stress alone or coupled with AR-antagonists. AR: adrenergic receptor, C: control, N. Saline: normal saline, All inhibitors: mice were treated with all AR-antagonists prior to stress, prazosin (alpha₁-AR antagonist), atipamezole (alpha₂-AR antagonist), propranolol (beta-AR antagonist). **P<0.01, ***P<0.001.</p

Stress-induced effect on TG homeostasis-related gene expression in the white adipose tissue.

<p>(A) Effect of restraint stress on the expression of genes involved in TG synthesis and lipolysis. (B) Effect of restraint stress on the expression of genes involved in TG metabolism and clearance. (C) Effect of restraint stress on the expression and activation of genes involved in TG hydrolysis. Comparisons were between controls and stress-exposed mice (alone or treated simultaneously with the AR-antagonists, prazosin, propranolol or atipamezole; black bars). DGAT1: Diacyl glycerol acyltransferase 1 (acyl coenzyme A (CoA), DGAT2: Diacyl glycerol acyltransferase 2, LPL: lipoprotein lipase, HSL: hormone sensitive lipase, ATGL/PNPLA2: adipose triglyceride lipase/patatin-like phospholipase domain containing 2, orphan nuclear receptor NR4A, AADAC: arylacetamide deacetylase, CD36: cluster of differentiation 36 or fatty acid transporter, CES3/TGH: carboxylesterase 3, MTTP: microsomal triglyceride transfer, PLIN5: perilipin 5. AR: adrenergic receptor, C: Control, Prazosin (alpha₁-AR antagonist), Atipamezole (alpha₂-AR antagonist), Propranolol (beta-AR antagonist), white adipose tissue (W.A.T.). Values are expressed as mean ± SE, n:5–6 per treatment group. Lanes in western blots correspond to one sample per treatment and represent one sample of three separate samples tested in different blots. *P<0.005, **P<0.01, ***P<0.001.</p

Metabolic map of osthole and its effect on lipids

<p>1. Osthole, a coumarin compound from plants, is a promising agent for the treatment of metabolic diseases, including hyperglycemia, fatty liver, and cancers. Studies indicate that the peroxisome proliferator-activated receptors (PPAR) α and γ are involved in the pharmacological effects of osthole. The <i>in vitro</i> and <i>in vivo</i> metabolism of osthole and its biological activity are not completely understood.</p> <p>2. In this study, ultra-performance chromatography electrospray ionization quadrupole time-of-flight mass spectrometry (UPLC–ESI–QTOFMS)-based metabolomics was used to determine the metabolic pathway of osthole and its influence on the levels of endogenous metabolites. Forty-one osthole metabolites, including 23 novel metabolites, were identified and structurally elucidated from its metabolism <i>in vitro</i> and <i>in vivo</i>. Recombinant cytochrome P450s (CYPs) screening showed that CYP3A4 and CYP3A5 were the primary enzymes contributing to osthole metabolism.</p> <p>3. More importantly, osthole was able to decrease the levels of lysophosphatidylethanolamine (LPE) and lysophosphatidylcholine (LPC) in the plasma, which explains in part its modulatory effects on metabolic diseases.</p> <p>4. This study gives the insights about the metabolic pathways of osthole <i>in vivo</i>, including hydroxylation, glucuronidation, and sulfation. Furthermore, the levels of the lipids regulated by osthole indicated its potential effects on adipogenesis. These data contribute to the understanding of the disposition and pharmacological activity of osthole <i>in vivo</i>.</p