Stimulation of cardiac cardiolipin biosynthesis by PPARalpha activation

The role of peroxisome proliferator-activated receptor alpha (PPARalpha)-stimulated phospholipase A2 (PLA2) in cardiac mitochondrial cardiolipin (CL) biosynthesis was examined in both in vivo and in vitro models. Treatment of rat heart H9c2 cells with clofibrate increased the expression and activity of 14 kDa PLA2 but did not affect the pool size of CL. Clofibrate treatment stimulated de novo CL biosynthesis via an increase in phosphatidylglycerolphosphate (PGP) synthase activity, accounting for the unaltered CL content. Cardiac PLA2, PGP synthase, and CDP-1,2-diacyl-sn-glycerol synthase (CDS-2) activities and CDS-2 mRNA levels were elevated in mice fed clofibrate for 14 days compared with controls. In PPARalpha-null mice, clofibrate feeding did not alter cardiac PLA2, PGP synthase activities, or CDS-2 activity and mRNA level, confirming that these enzymes are regulated by PPARalpha activation. In contrast to mouse heart, clofibrate treatment did not affect the activity or mRNA levels of CDS-2 in H9c2 cells, indicating that CDS-2 is regulated differently in rat heart H9c2 cells in vitro and in mouse heart in vivo. These results clearly indicate that cardiac CL de novo biosynthesis is stimulated by PPARalpha activation in responsive rodent models and that CDS-2 is an example of an enzyme that exhibits alternative regulation in vivo and in cultured cell lines. This study is the first to demonstrate that CL de novo biosynthesis is regulated by PPARalpha activation.     

Chondrocyte cell death mediated by reactive oxygen species-dependent activation of PKC-betaI

Signals generated by the extracellular matrix (ECM) promote cell survival. We have shown that chondrocytes detached from their native ECM and plated without serum at low density on poly-L-lysine undergo significant cell death that is associated with the production of reactive oxygen species (ROS). No cell death or ROS production was observed when cells were plated on fibronectin under the same conditions. Cell death on poly-L-lysine could be completely inhibited with the addition of either antioxidants or inhibitors of specific protein kinase C (PKC) isoforms including PKC-I. PKC-I was noted to translocate from the cytosol to the particulate membrane after plating on poly-L-lysine, and this translocation was inhibited by the addition of an antioxidant. Time-course analyses implicated endogenous ROS production as a secondary messenger leading to PKC-I activation and subsequent chondrocyte cell death. Cell survival on poly-L-lysine was significantly improved in the presence of oligomycin or DIDS, suggesting that ROS production occurred via complex V of the electron transport chain of the mitochondria and that ROS were released to the cytosol via voltage-dependent anion channels. Together, these results represent a novel mechanism by which ROS can initiate cell death through the activation of PKC-I. articular cartilage; osteoarthritis; cell signaling; fibronectin     

PPARalpha activation increases triglyceride mass and adipose differentiation-related protein in hepatocytes

Adipose differentiation-related protein (ADRP) is a lipid droplet-associated protein that is expressed in various tissues. In mice treated with the peroxisome proliferator-activated receptor alpha (PPARalpha) agonist Wy14,643 (Wy), hepatic mRNA and protein levels of ADRP as well as hepatic triglyceride content increased. Also in primary mouse hepatocytes, Wy increased ADRP expression and intracellular triglyceride mass. The triglyceride mass increased in spite of unchanged triglyceride biosynthesis and increased palmitic acid oxidation. However, Wy incubation decreased the secretion of newly synthesized triglycerides, whereas apolipoprotein B secretion increased. Thus, decreased availability of triglycerides for VLDL assembly could help to explain the cellular accumulation of triglycerides after Wy treatment. We hypothesized that this effect could be mediated by increased ADRP expression. Similar to PPARalpha activation, adenovirus-mediated ADRP overexpression in mouse hepatocytes enhanced cellular triglyceride mass and decreased the secretion of newly synthesized triglycerides. In ADRP-overexpressing cells, Wy incubation resulted in a further decrease in triglyceride secretion. This effect of Wy was not attributable to decreased cellular triglycerides after increased fatty acid oxidation because the triglyceride mass in Wy-treated ADRP-overexpressing cells was unchanged. In summary, PPARalpha activation prevents the availability of triglycerides for VLDL assembly and increases hepatic triglyceride content in part by increasing the expression of ADRP.     

PPARalpha activators upregulate eNOS activity and inhibit cytokine-induced NF-kappaB activation through AMP-activated protein kinase activation

Endothelium-derived NO is an important mediator of vascular protection and adhesion molecule expression on the endothelial cell surface is critical for leukocyte recruitment to atherosclerotic lesions. We hypothesized that AMP-activated protein kinase (AMPK) activity is a down-stream mediator of the beneficial effects of PPARalpha activators on vascular endothelial cells. Treatment of human umbilical vein endothelial cells (HUVEC) with fenofibrate or WY14643 resulted in transient activation of AMPK, as monitored by phosphorylation of AMPK and its down-stream target, acetyl-CoA carboxylase. Fenofibrate caused phosphorylation of Akt and eNOS, leading to increased production of NO, and also caused inhibition of cytokine-induced NF-kappaB activation, leading to suppression of expression of adhesion molecule genes. Significant decreases in eNOS activity and NO production in response to fenofibrate were observed in cells treated with AMPK siRNA or with AraA, a pharmacological inhibitor of AMPK. The attenuation of fenofibrate-induced inhibition of NF-kappaB activation was observed in mouse endothelial (SVEC4) cells treated with AMPK siRNA or with AraA. We demonstrated that TNFalpha stimulates IkappaB-alpha phosphorylation through induction of IKK activity, and that fenofibrate inhibits IKK activity and TNFalpha-induced IkappaB-alpha phosphorylation. Our findings suggest that the beneficial effects of PPARalpha activators on endothelial cells such as inhibition of diabetic microangiopathy might be attributed to the induction of AMPK activation beyond its lipid-lowering actions.     

Reactive oxygen species-dependent RhoA activation mediates collagen synthesis in hyperoxic lung fibrosis

Lung fibrosis is an ultimate consequence of pulmonary oxygen toxicity in human and animal models. Excessive production and deposition of extracellular matrix proteins, e.g., collagen-I, is the most important feature of pulmonary fibrosis in hyperoxia-induced lung injury. In this study, we investigated the roles of RhoA and reactive oxygen species (ROS) in collagen-I synthesis in hyperoxic lung fibroblasts and in a mouse model of oxygen toxicity. Exposure of human lung fibroblasts to hyperoxia resulted in RhoA activation and an increase in collagen-I synthesis and cell proliferation. Inhibition of RhoA by C3 transferase CT-04, dominant-negative RhoA mutant T19N, or RhoA siRNA prevented hyperoxia-induced collagen-I synthesis. The constitutively active RhoA mutant Q63L mimicked the effect of hyperoxia on collagen-I expression. Moreover, the Rho kinase inhibitor Y27632 inhibited collagen-I synthesis in hyperoxic lung fibroblasts and fibrosis in mouse lungs after oxygen toxicity. Furthermore, the ROS scavenger tiron attenuated hyperoxia-induced increases in RhoA activation and collagen-I synthesis in lung fibroblasts and mouse lungs after oxygen toxicity. More importantly, we found that hyperoxia induced separation of guanine nucleotide dissociation inhibitor (GDI) from RhoA in lung fibroblasts and mouse lungs. Further, tiron prevented the separation of GDI from RhoA in hyperoxic lung fibroblasts and mouse lungs with oxygen toxicity. Together, these results indicate that ROS-induced separation of GDI from RhoA leads to RhoA activation with oxygen toxicity. ROS-dependent RhoA activation is responsible for the increase in collagen-I synthesis in hyperoxic lung fibroblasts and mouse lungs.     

Inward remodeling of resistance arteries requires reactive oxygen species-dependent activation of matrix metalloproteinases

Inward eutrophic remodeling is the most prevalent structural change of resistance arteries in hypertension. Sympathetic and angiotensin (ANG)-induced vasoconstriction has been associated with hypertension and with the production of matrix metalloproteinases (MMPs) and ROS. Therefore, we hypothesize that prolonged exposure to norepinephrine (NE) and ANG II induces arteriolar inward remodeling dependent on the activation of MMPs and the production of ROS. This hypothesis was tested on rat cremaster arterioles that were isolated, cannulated, pressurized, and exposed to either NE (10(-5.5) mol/l) + ANG II (10(-7) mol/l) or vehicle (control) for 4 h. The prolonged exposure to NE + ANG II induced inward remodeling, as evidenced by the reduced maximal arteriolar passive diameter observed after versus before exposure to the vasoconstrictor agonists. NE + ANG II also increased the arteriolar expression and activity of MMP-2 and the production of ROS as determined, respectively, by real-time RT-PCR, gel and in situ zymography, and the use of ROS-sensitive dyes with multiphoton microscopy. Inhibition of MMP activation (with GM-6001) or ROS production (with apocynin or tempol) prevented the NE + ANG II-induced inward remodeling. Inhibition of ROS production prevented the activation of MMPs and the remodeling process, whereas inhibition of MMP activation did not affect ROS production. These results indicate that prolonged stimulation of resistance arterioles with NE + ANG II induces a ROS-dependent activation of MMPs necessary for the development of arteriolar inward remodeling. These mechanisms may contribute to the structural narrowing of resistance vessels in hypertension.     

Impaired Ras membrane association and activation in PPARalpha knockout mice after partial hepatectomy

Liver regeneration after partial hepatectomy (PH) involves several signaling mechanisms including activation of the small GTPases Ras and RhoA in response to mitogens leading to DNA synthesis and cell proliferation. Peroxisome proliferator-activated receptor-alpha (PPARalpha) regulates the expression of several key enzymes in isoprenoid synthesis, which are key events for membrane association of Ras and RhoA. Thus the role of PPARalpha in cell proliferation after PH was tested. After PH, an increase in PPARalpha DNA binding was observed in wild-type mice, correlating with an increase in the PPARalpha-regulated enzyme acyl-CoA oxidase. In addition, the PPARalpha-regulated genes farnesyl pyrophosphate synthase and 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) synthase were significantly increased in wild-type mice. However, these increases were not observed in PPARalpha knockout (PPARalpha -/-) mice. The peak in DNA synthesis observed 42 h after PH was reduced by approximately 60% in PPARalpha -/- mice, despite increases in TNF-alpha and IL-1. Also, under these conditions, membrane association of Ras was high in wild-type mice after PH but was impaired in PPARalpha -/- mice. Accordingly, Ras was significantly elevated in the cytosol in PPARalpha -/- mice. This observation correlated with lower levels of active GTP-bound Ras after PH in PPARalpha -/- mice compared with wild-type mice. Similar observations were made for RhoA. Moreover, deletion of PPARalpha blunted the activation of cyclin-dependent kinase (cdk)2/cyclin E and cdk4/cyclin D complexes. Collectively, these results support the hypothesis that PPARalpha is necessary for cell cycle progression in regenerating mouse liver via mechanisms involving prenylation of small GTPases Ras and RhoA.     

PPARalpha activation elevates blood pressure and does not correct glucocorticoid-induced insulin resistance in humans

Fibrates, activators of the nuclear receptor PPARalpha, improve dyslipidemia, but their effects on insulin resistance and vascular disease are unresolved. To test the hypothesis that PPARalpha activation improves insulin resistance and vascular function, we determined the effects of fenofibrate in healthy adults with insulin resistance induced by short-term glucocorticoid administration. Eighteen normal-weight subjects were studied in four stages: at baseline, after 21 days of fenofibrate (160 mg/day) alone, after 3 days of dexamethasone (8 mg/day) added to fenofibrate, and after 3 days of dexamethasone added to placebo (dexamethasone alone). Dexamethasone alone caused hyperinsulinemia, increased glucose, decreased glucose disposal, and reduced insulin-induced suppression of hepatic glucose production as determined by hyperinsulinemic euglycemic clamp and increased systolic blood pressure as determined by ambulatory monitoring, features associated with an insulin-resistant state. Fenofibrate improved fasting LDL and total cholesterol in the setting of dexamethasone treatment but had no significant effect on levels of insulin or glucose, insulin-stimulated glucose disposal, or insulin suppression of glucose production during clamps, or ambulatory monitored blood pressure. In the absence of dexamethasone, fenofibrate lowered fasting triglycerides and cholesterol but unexpectedly increased systolic blood pressure by ambulatory monitoring. These data suggest that PPARalpha activation in humans does not correct insulin resistance induced by glucocorticoids and may adversely affect blood pressure.     

Circadian expression of FGF21 is induced by PPARalpha activation in the mouse liver

Peroxisome proliferator-activated receptor alpha (PPARalpha) is a nuclear receptor that regulates the expression of genes associated with lipid metabolism. Recent studies have suggested that the expression of PPARalpha-dependent fibroblast growth factor 21 (FGF21) plays important roles in adaptation to fasting, such as lipolysis and ketogenesis. We found that a nighttime injection of bezafibrate, a ligand of PPARalpha, effectively induced FGF21 expression, whereas a daytime injection did not affect it. Furthermore, bezafibrate-induced circadian FGF21 expression was abolished in PPARalpha-deficient mice. These observations suggest that bezafibrate-induced circadian FGF21 expression is due to circadian variations in the responsiveness of the PPARalpha system in the liver.     

An afferent vagal nerve pathway links hepatic PPARalpha activation to glucocorticoid-induced insulin resistance and hypertension

Glucocorticoid excess causes insulin resistance and hypertension. Hepatic expression of PPARalpha (Ppara) is required for glucocorticoid-induced insulin resistance. Here we demonstrate that afferent fibers of the vagus nerve interface with hepatic Ppara expression to disrupt blood pressure and glucose homeostasis in response to glucocorticoids. Selective hepatic vagotomy decreased hyperglycemia, hyperinsulinemia, hepatic insulin resistance, Ppara expression, and phosphoenolpyruvate carboxykinase (PEPCK) enzyme activity in dexamethasone-treated Ppara(+/+) mice. Selective vagotomy also decreased blood pressure, adrenergic tone, renin activity, and urinary sodium retention in these mice. Hepatic reconstitution of Ppara in nondiabetic, normotensive dexamethasone-treated PPARalpha null mice increased glucose, insulin, hepatic PEPCK enzyme activity, blood pressure, and renin activity in sham-operated animals but not hepatic-vagotomized animals. Disruption of vagal afferent fibers by chemical or surgical means prevented glucocorticoid-induced metabolic derangements. We conclude that a dynamic interaction between hepatic Ppara expression and a vagal afferent pathway is essential for glucocorticoid induction of diabetes and hypertension.     

Pharmacology of PPARalpha, PPARgamma and dual PPARalpha/gamma agonists in clinical development

Cardiovascular diseases (CVD) remain the leading cause of mortality in the western societies. Several risk factors predispose to CVD including diabetes, obesity, insulin resistance, dyslipidemia and hypertension. Various pharmacological therapies have been developed to control the risk factors associated to CVD. Fibrates are able to correct dyslipidemia, therefore decreasing CVD risk. Thiazolidinediones (TZD) or glitazones by increasing insulin sensitivity decrease plasma glucose levels in diabetic patients. Both fibrates and TZD activate the peroxisome proliferator-activated receptors (PPARs), a family of nuclear receptors that play a central role in the control of lipid and glucose metabolism. In this review, we will discuss the mode of action of fibrates and TZD and we will present an overview on PPAR ligands under development.     

The circulating metabolic regulator FGF21 is induced by prolonged fasting and PPARalpha activation in man

FGF21 is a critical metabolic regulator, pivotal for fasting adaptation and directly regulated by PPARα in rodents. However, the physiological role of FGF21 in man is not yet defined and was investigated in our study. Serum FGF21 varied 250-fold among 76 healthy individuals and did not relate to age, gender, body mass index (BMI), serum lipids, or plasma glucose. FGF21 levels had no diurnal variation and were unrelated to bile acid or cholesterol synthesis. Ketosis induced by a 2 day fast or feeding a ketogenic diet (KD) did not influence FGF21 levels, whereas a 74% increase occurred after 7 days of fasting. Hypertriglyceridemic nondiabetic patients had 2-fold elevated FGF21 levels, which were further increased by 28% during fenofibrate treatment. FGF21 circulates in human plasma and increases by extreme fasting and PPARα activation. The wide interindividual variation and the induction of ketogenesis independent of FGF21 levels indicate that the physiological role of FGF21 in humans may differ from that in mice.     

Synergistic activation of UCP-3 expression in cultured fetal rat brown adipocytes by PPARalpha and PPARgamma ligands

Rat brown adipocytes express mRNAs for Uncoupling Proteins (UCP) 1, 2 and 3 and the Peroxisome Proliferator Activated Receptors (PPAR) alpha and gamma. We have examined the effects of selective PPARalpha or -gamma activation on changes in UCP-1 and UCP-3 mRNA levels in cultured fetal rat brown adipocytes (FBA). Rosiglitazone (1.0 microM), a selective PPARgamma agonist, elicited 5- and 3-fold increases in UCP-1 and UCP-3, respectively. The PPARalpha ligand, Wy14643 (10.0 microM) increased UCP-3 tenfold, but decreased UCP-1. A synergistic effect on UCP-3 expression (30-fold increase; P < 0. 05) was observed when FBA were exposed to a combination of Wy14643 (10.0 microM) and rosiglitazone (10.0 microM). Thus, activation of PPARgamma increases UCP-1 and UCP-3 levels which are differentially regulated by PPARalpha. A synergistic interaction occurs between PPARalpha and PPARgamma in the regulation of UCP-3 in FBA, probably via co-activator recruitment, suppression of co-repressor proteins or through a direct interaction at the level of the PPRE.