Paraoxonase (PON1) deficiency is associated with increased macrophage oxidative stress: studies in PON1-knockout mice

Human serum paraoxonase (PON1), an HDL-associated esterase, protects lipoproteins against oxidation, probably by hydrolyzing specific lipid peroxides. As arterial macrophages play a key role in oxidative stress in early atherogenesis, the aim of the present study was to examine the effect of PON1 on macrophage oxidative stress. For this purpose we used mouse arterial and peritoneal macrophages (MPM) that were harvested from two populations of PON1 knockout (KO) mice: one on the genetic background of C57BL/6J (PON1(0)) and the other one on the genetic background of apolipoproteinE KO (PON1(0)/E(0)). Serum and LDL, but not HDL, lipids peroxidation was increased in PON1(0), compared to C57BL/6J mice, by 84% and by 220%, respectively. Increased oxidative stress was shown in peritoneal and in arterial macrophages derived from either PON1(0) or PON1(0)/E(0) mice, compared to their appropriate controls. Macrophage oxidative stress was expressed by increased lipid peroxides content in MPM from PON1(0) and from PON1(0)/E(0) mice by 48% and by 80%, respectively, and by decreased reduced glutathione (GSH) content, compared to the appropriate controls. Furthermore, increased capacity of MPM from PON1(0) and PON1(0)/E(0) mice to oxidize LDL (by 40% and by 19%, respectively) and to release superoxide anions was observed. In accordance with these results, PON1(0) mice MPM exhibited 130% increased translocation of the cytosolic p47phox component of NADPH-oxidase to the macrophage plasma membrane, suggesting increased activation of macrophage NADPH-oxidase in PON1(0) mice, compared to control mice MPM. The increase in oxidative stress in PON1-deficient mice was observed despite the presence of the two other members of the PON gene family. PON2 and PON3 activities and mRNA expression were both found to be present in PON1-deficient mice MPM. Upon incubation of PON1(0)/E(0) derived macrophages with human PON1 (7.5 arylesterase units/ml), cellular peroxides content was decreased by 18%, macrophage superoxide anion release was decreased by 33%, and macrophage-mediated oxidation of LDL was reduced by 22%. Finally, a 42% increase in the atherosclerotic lesion area was observed in PON1(0)/E(0) mice, in comparison to E(0) mice under regular chow diet. We thus concluded that PON1 can directly reduce oxidative stress in macrophages and in serum, and that PON1-deficiency results in increased oxidative stress not only in serum, but also in macrophages, a phenomenon that can contribute to the accelerated atherosclerosis shown in PON1-deficient mice.     

Paraoxonases 1, 2, and 3, oxidative stress, and macrophage foam cell formation during atherosclerosis development

Paraoxonases PON1 and PON3, which are both associated in serum with HDL, protect the serum lipids from oxidation, probably as a result of their ability to hydrolyze specific oxidized lipids. The activity of HDL-associated PON1 seems to involve an activity (phospholipase A2-like activity, peroxidase-like activity, lactonase activity) which produces LPC. To study the possible role of PON1 in macrophage foam cell formation and atherogenesis we used macrophages from control mice, from PON1 knockout mice, and from PON1 transgenic mice. Furthermore, we analyzed PON1-treated macrophages and PON1-transfected cells to demonstrate the contribution of PON1 to the attenuation of macrophage cholesterol and oxidized lipid accumulation and foam cell formation. PON1 was shown to inhibit cholesterol influx [by reducing the formation of oxidized LDL (Ox-LDL), increasing the breakdown of specific oxidized lipids in Ox-LDL, and decreasing macrophage uptake of Ox-LDL]. PON1 also inhibits cholesterol biosynthesis and stimulates HDL-mediated cholesterol efflux from macrophages. PON2 and PON3 protect against oxidative stress, with PON2 acting mainly at the cellular level. Whereas serum PON1 and PON3 were inactivated under oxidative stress, macrophage PON2 expression and activity were increased under oxidative stress, probably as a compensatory mechanism against oxidative stress. Intervention to increase the paraoxonases (cellular and humoral) by dietary or pharmacological means can reduce macrophage foam cell formation and attenuate atherosclerosis development.     

Impaired hepatic insulin signalling in PON2-deficient mice: a novel role for the PON2/apoE axis on the macrophage inflammatory response

Hepatic glucose metabolism is strongly influenced by oxidative stress and pro-inflammatory stimuli. PON2 (paraoxonase 2), an enzyme with undefined antioxidant properties, protects against atherosclerosis. PON2-deficient (PON2-def) mice have elevated hepatic oxidative stress coupled with an exacerbated inflammatory response from PON2-deficient macrophages. In the present paper, we demonstrate that PON2 deficiency is associated with inhibitory insulin-mediated phosphorylation of hepatic IRS-1 (insulin receptor substrate-1). Unexpectedly, we observed a marked improvement in the hepatic IRS-1 phosphorylation state in PON2-def/apoE (apolipoprotein E)(-/-) mice, relative to apoE(-/-) mice. Factors secreted from activated macrophage cultures derived from PON2-def and PON2-def/apoE(-/-) mice are sufficient to modulate insulin signalling in cultured hepatocytes in a manner similar to that observed in vivo. We show that the protective effect on insulin signalling in PON2-def/apoE(-/-) mice is directly associated with altered production of macrophage pro-inflammatory mediators, but not elevated intracellular oxidative stress levels. We further present evidence that modulation of the macrophage inflammatory response in PON2-def/apoE(-/-) mice is mediated by a shift in the balance of NO and ONOO(-) (peroxynitrite) formation. Our results demonstrate that PON2 plays an important role in hepatic insulin signalling and underscores the influence of macrophage-mediated inflammatory response on hepatic insulin sensitivity.     

Oxidized phospholipids induce changes in hepatic paraoxonase and ApoJ but not monocyte chemoattractant protein-1 via interleukin-6

In this study, we tested if interleukin-6 (IL-6) plays a role in mediating the effects of oxidized phospholipids (OXPL). Treatment of HepG2 cells with oxidized 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphoryl choline (OX-PAPC), or biologically active lipids present in mildly oxidized low density lipoprotein, increased apolipoprotein J (apoJ), and decreased paraoxonase (PON) mRNA levels. Antibodies to IL-6 blocked these changes. IL-6 treatment in the absence of OXPL produced the same pattern of mRNA changes observed with OXPL treatment alone. In vivo, OX-PAPC injected into C57BL/6J mice resulted in a marked reduction in PON activity and an increase in apoJ levels in plasma after 16 h. Injection of OX-PAPC into IL-6-deficient C57BL/6J mice (IL-6 -/-) did not alter either PON activity or apoJ levels. We then tested if other mechanisms involved in fatty streak formation depended upon IL-6. Antibody to IL-6 had no effect on OX-PAPC-induced secretion of MCP-1 by endothelial cells nor on MCP-1 mRNA expression in HepG2 cells. C57BL/6J and IL-6 -/- mice fed an atherogenic diet both demonstrated markedly reduced plasma PON activities and the IL-6 -/- mice developed fatty streaks to a greater degree than wild-type mice. We conclude that IL-6 is critical to short term but not long term regulation of PON and that IL-6 is not required for OXPL regulation of MCP-1.     

Paraoxonase, a cardioprotective enzyme: continuing issues

PURPOSE OF REVIEW: The paraoxonase family consists of three members (PON1, PON2 and PON3) that share structural properties and enzymatic activities, among which is the ability to hydrolyze oxidized lipids in LDL. The exact function of the different family members is not clear although the conservation among the individual family members across species suggests a strong evolutionary pressure to preserve these functional differences. The purpose of this review is to highlight several problems with respect to the mechanism of action of paraoxonase and differences between the family members that merit further study. RECENT FINDINGS: PON1 transgenic mice are at lower risk for atherosclerosis, which is consistent with PON1 gene knockout studies in mice and human genetic polymorphism studies. The exact mechanism by which paraoxonase is cardioprotective is not clear, although it is likely to be related to its antioxidant properties especially on LDL. PON1 levels are influenced by a variety of environmental factors, including statins and cytokines. The preferential association of PON1 with HDL is mediated in part by its signal peptide and by desorption from the plasma membrane of expressing cells by HDL or phospholipid. Apolipoprotein A-I is not necessary for PON1 association with HDL, but its activity is stabilized in the presence of the apolipoprotein. Only in the absence of both lecithin cholesterol acyltransferase and apolipoprotein E is paraoxonase associated with non-HDL lipoproteins. The displacement of paraoxonase by serum amyloid A may explain in part the proinflammatory nature of HDL in the acute phase. The mechanism by which PON3 associates with HDL has not been studied. In addition to the ability to hydrolyze oxidized lipids in LDL, paraoxonase also alters the oxidative state of macrophages. Exogenous PON1 is able to reverse the oxidative stress in macrophages in aged apolipoprotein E deficient and PON1 deficient mice. The increase in oxidative stress in macrophages from PON1 deficient mice occurs despite the expression of PON2 and PON3 in macrophages. PON1 has recently been shown to contain phospholipase A2 activity, with the subsequent release of lysophosphatidylcholine that influences macrophage cholesterol biosynthesis. SUMMARY: PON1 mass and activity in the plasma significantly influence the risk of developing cardiovascular disease. This is likely mediated by its antioxidation properties on LDL and/or macrophages. The precise mechanism by which this HDL associated protein prevents or attenuates oxidation of LDL and the oxidative stress of macrophages remains to be clarified. The role of PON2 and PON3 in atherosclerosis and their antioxidant properties with respect to LDL and macrophages also merit further investigation.     

Lack of adiponectin promotes formation of cholesterol gallstones in mice

Plasma adiponectin levels are reduced in obese people, and hypoadiponectinemia is recently reported to associate with cholesterol gallstone formation in human. The aim of this study was to examine the role of adiponectin in gallstone formation using adiponectin knockout mice. We analyzed male knockout and C57BL6J mice fed normal or lithogenic diet for 6 weeks. On lithogenic diet, the prevalence rate of gallstone was significantly greater in knockout mice than C57BL6J mice. The molar percentages of β and ω-muricholic acid were significantly higher and hepatic sterol 12α-hydroxylase expression (cyp8b1) was significantly lower in knockout mice than C57BL6J mice fed normal diet. The bile apolipoprotein A-I protein levels were decreased in knockout mice. Histological examination showed gallbladder wall thickening and accumulation of glycoprotein in the gallbladder of knockout mice. Gallbladder phospholipase A2-IVA expression was significantly higher in knockout mice than in C57BL6J mice fed lithogenic diet. Our results indicate that lack of adiponectin promotes gallstone formation in mice.     

Effect of the walnut polyphenol fraction on oxidative stress in type 2 diabetes mice

We examined the in vivo antioxidative effect of a polyphenol-rich walnut extract on oxidative stress in mice with type 2 diabetes. C57BL/KsJ-db/db mice were used as an accelerated oxidative animal model. The oral administration of the walnut polyphenol fraction at 200 mg/kg body weight for 4 weeks caused a significant decrease in the level of urinary 8-hydroxy-2'-deoxyguanosin, which is an in vivo marker of oxidative stress. These results imply that walnut polyphenols have both in vitro and in vivo antioxidant effects.     

Paraoxonases (PONs) 1, 2, and 3 are expressed in human and mouse gastrointestinal tract and in Caco-2 cell line: selective secretion of PON1 and PON2

The paraoxonase (PON) family contains three genes (PON1/2/3) that are believed to be involved in the protection against oxidative stress. PON1 and PON3 are circulating in serum attached to high-density lipoprotein fraction (HDL), whereas PON2 is ubiquitously expressed. The intestine is the second major organ that synthesizes lipoproteins; therefore, we examined PON mRNA expression and protein levels in gastrointestinal biopsies from humans, from C57BL6 mice, and from Caco-2 cells, a colon carcinoma-derived cell line that exhibits properties of intestinal epithelium at differentiation. PON 1/2/3 mRNA and proteins were present in human biopsies with variable expression among different gastrointestinal segments. Only PON2 and PON3 were present in mice. All PON mRNA, proteins, and enzymatic activities were present in Caco-2 cells. Oxidation of CaCo-2 cells with ferrum ascorbate had no significant effect on PON mRNA expression, but it increased paraoxonase and lactonase activity, whereas statinase activity was decreased. We showed polarized secretion of PON1 (basolateral) and PON2 (apical) into Caco-2 culture medium, raising the possibility that intestine is capable of producing and releasing PON1 and PON3 to the circulation, whereas PON2 is released at the brush-border membrane to intestinal lumen where it may perform another yet unclear function.     

Oxidative stress induced by high-glucose diet in liver of C57BL/6J mice and its underlying mechanism

High glycemic index diet can induce multiple diseases. Many research indicated that oxidative stress played important role in many pathological conditions. However, the impact of gene expression and dietary habit on oxidation process are still less clear. We used high-glucose diet to feed C57BL/6J mice for 4 weeks, measured the redox status, physiological and biochemical changes related to diabetes and consequence of metabolic syndrome (nonalcoholic fatty liver, cardiovascular disease), and detected the expressions of 14,446 genes in liver of C57BL/6J mice with DNA microarray. The results showed high-glucose diet induced elevated fatty acid accumulation in liver, insulin resistance index and higher weight in C57BL/6J mice, which indicated high-glucose diet caused to the initiation and development of diabetes and consequence of metabolic syndrome. The results also showed high-glucose diet induced oxidative stress in liver of C57BL/6J mice, which might the cause of initiation and development of diabetes and consequence of metabolic syndrome. Microarray analysis found expressions of genes related to thiol redox, fatty acid oxidation in peroxisome and cytochrome P450 were significantly changed, indicating system in which non-enzyme antioxidant capacity was impaired and sources from which reactive oxygen species (ROS) generated, which revealed the molecular mechanism of oxidative stress induced by high-glucose diet. We validated our microarray findings by conducting real-time RT–PCR assays on selected genes.     

Macrophage paraoxonase 1 (PON1) binding sites

Paraoxonase 1 (PON1), an HDL-associated esterase, is known to possess anti-oxidant and anti-atherogenic properties. PON1 was shown to protect macrophages from oxidative stress, to inhibit macrophage cholesterol biosynthesis, and to stimulate HDL-mediated cholesterol efflux from the cells. The aim of the present study was to characterize macrophage PON1 binding sites which could be responsible for the above anti-atherogenic activities.Incubation of FITC-labeled recombinant PON1 with J774 A.1 macrophage-like cell line at 37 °C, resulted in cellular binding and internalization of PON1, leading to PON1 localization in the cell’s cytoplasm compartment. In order to determine whether PON1 uptake is mediated via a specific binding to the macrophage, FITC-labeled recombinant PON1 was incubated with macrophages at 4 °C, followed by cell membranes separation. Macrophage membrane fluorescence was shown to be directly and dose-dependently related to the labeled PON1 concentration. Furthermore, binding assays performed at 4 and at 37 °C, using labeled and non-labeled recombinant PON1 (for competitive inhibition), demonstrated a dose-dependent significant 30% decrement in labeled PON1 binding to the macrophages, by the non-labeled PON1. Similarly, binding assays, using labeled PON1 and non-labeled HDL (the natural carrier of PON1 in the circulation) indicated that HDL decreased the binding of labeled PON1 to macrophages by 25%. Unlike HDL, LDL had no effect on labeled PON1 binding to macrophages. Finally, HDL were pre incubated without or with PON1 or apolipoprotein AI (apoAI) antibodies, in order to block PON1 or apoAI ability to bind to the cells. HDL incubation with antibody to PON1 or to apoAI significantly decreased HDL ability to inhibit macrophages-mediated LDL oxidation (by 32% or by 25%, respectively). A similar trend was also observed for HDL-mediated cholesterol efflux from macrophages, with an inhibitory effect of 35% or 19%, respectively. These results suggest that blocking HDL binding to macrophages through its apo A-I, and more so, via its PON1, results in the attenuation of HDL-PON1 biological activities.In conclusion, PON1 specifically binds to macrophage binding sites, leading to anti-atherogenic effects. Macrophage PON1 binding sites may thus be a target for future cardio protection therapy.     

Paraoxonases and cardiovascular diseases: pharmacological and nutritional influences

PURPOSE OF REVIEW: To summarize the new articles published in the last year on paraoxonases, including their expression in cardiovascular diseases, and regulation by pharmacological and nutritional means. RECENT FINDINGS: The elucidation of the crystal structure of the paraoxonase 1 (PON1) gene, obtained by directed evolution, shows that it consists of a six-bladed beta-propeller with a unique active site. PON1 is present in HDL but also in lipoprotein-deficient serum, in VLDL and in chylomicrons. PON1 protects lipids in lipoproteins, in macrophages and in erythrocytes from oxidation. Cellular PON2 and PON3 were also shown to reduce oxidative stress. Beyond its antioxidative properties, PON1 possesses additional antiatherogenic properties against macrophage foam cell formation: attenuation of cholesterol and oxidized lipids influx, inhibition of macrophage cholesterol biosynthesis and stimulation of macrophage cholesterol efflux. The PON1 gene is regulated by Sp1 and protein kinase C, whereas the PON2 gene in macrophages is regulated by nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. PON1 activity and mass are both reduced in cardiovascular diseases and the hypocholesterolemic drugs, statins, increase serum PON1 activity (by reducing oxidative stress, or by upregulating hepatic PON1 expression). Expression of cellular PON2, like PON1, was upregulated by statins. Nutritional antioxidants, such as polyphenols, increase PON1 mRNA expression and activity, by an aryl hydrocarbon receptor-dependent mechanism. SUMMARY: The elucidation of PON1 structure and its active center has enabled a better understanding of its mechanism of action, including its physio-pathological substrate(s). Some drugs and nutrients including dietary antioxidants and polyphenols considerably increase the activities of paraoxonases which, in turn, can reduce oxidative stress and atherosclerosis development.     

Activation of PAR2 promotes high-fat diet-induced renal injury by inducing oxidative stress and inflammation

A high-fat diet (HFD) is a major risk factor for chronic kidney disease. Although HFD promotes renal injury, characterized by increased inflammation and oxidative stress leading to fibrosis, the underlying mechanism remains elusive. Here, we investigated the role and mechanism of protease-activating receptor 2 (PAR2) activation during HFD-induced renal injury in C57/BL6 mice. HFD for 16 weeks resulted in kidney injury, manifested by increased blood levels of blood urea nitrogen, increased levels of oxidative stress with inflammation, and structural changes in the kidney tubules. HFD-fed kidneys showed elevated PAR2 expression level in the tubular epithelial region. To elucidate the role of PAR2, PAR2 knockout mice and their littermates were administered HFD. PAR2 deficient kidneys showed reduced extent of renal injury. PAR2 deficient kidneys showed significantly decreased levels of inflammatory gene expression and macrophage infiltration, followed by reduced accumulation of extracellular matrix proteins. Using NRK52E kidney epithelial cells, we further elucidated the mechanism and role of PAR2 activation during renal injury. Palmitate treatment increased PAR2 expression level in NRK52E cells and scavenging of oxidative stress blocked PAR2 expression. Under palmitate-treated conditions, PAR2 agonist-induced NF-κB activation level was higher with increased chemokine expression level in the cells. These changes were attenuated by the depletion of oxidative stress. Taken together, our results suggest that HFD-induced PAR2 activation is associated with increased levels of renal oxidative stress, inflammatory response, and fibrosis.     

A biphasic U-shape effect of cellular oxidative stress on the macrophage anti-oxidant paraoxonase 2 (PON2) enzymatic activity

Expression of macrophage paraoxonase 2 (PON2), a cellular lactonase with anti-oxidant and anti-atherogenic properties, was shown to be upregulated under high oxidative stress. The aim of the present study was to analyze the relationship between the extent of cellular oxidative stress in J774A.1 macrophage and PON2 lactonase activity under various levels of oxidation, obtained by cell incubation with either anti-oxidants or oxidants. PON2 activity exhibited a U-shape response curve. In the oxidative stress range below that of control untreated cells, PON2 activity decreased upon increasing macrophage oxidative state, whereas in the range over that of control untreated cells, PON2 activity increased. The biphasic effect of oxidative stress on macrophage PON2 activity could be related to PON2 inactivation (decreased enzymatic activity) under oxidative stress induction at its low range, whereas at high range of oxidative stress, macrophage anti-oxidant compensatory mechanism up-regulates PON2 (increased protein expression), in order to cope with oxidative burden.     

Macrophages from alpha 7 nicotinic acetylcholine receptor knockout mice demonstrate increased cholesterol accumulation and decreased cellular paraoxonase expression: A possible link between the nervous system and atherosclerosis development

Objective

The parasympathetic nervous system regulates inflammation in peripheral tissues through a pathway termed the “cholinergic anti-inflammatory reflex” (CAIR). Mice deficient in the alpha 7 nicotinic acetylcholine receptor (α7^−/−) have an impaired CAIR due to decreased signaling through this pathway. The purpose of this study was to determine if the increased inflammation in α7^−/− mice is associated with enhanced serum and macrophage atherogenicity.

Methods

We measured serum markers of inflammation and oxidative stress, and macrophage atherogenicity in mouse peritoneal macrophages harvested from α7^−/− mice on the background of C57BL/6 mice, as well as on the background of the atherosclerotic Apolipoprotein E-deficient (ApoE^−/−) mice.

Results

α7-Deficiency had no significant effects on serum cholesterol, or on markers of serum oxidative stress (TBARS and paraoxonase1 activities). However, α7-deficiency significantly increased serum CRP and IL-6 (p < 0.05) levels in atherosclerotic mice, confirming an anti-inflammatory role for the α7 receptor. Macrophage cholesterol mass was increased by 25% in both normal and atherosclerotic mice in the absence of the α7 receptor (p < 0.05). This was accompanied by conditional increases in oxidized LDL uptake and in macrophage total peroxide levels. Furthermore, α7-deficiency reduced macrophage paraoxonase2 mRNA and activity by 50-100% in normal and atherosclerotic mice (p < 0.05 for each), indicating a reduction in macrophage anti-oxidant capacity in the α7^−/− mice.

Conclusion

The above results suggest an anti-atherogenic role for the macrophage α7nAchr, through a mechanism that involves attenuated macrophage oxidative stress and decreased uptake of oxidized LDL.     

S-Glutathionylation regulates HDL-associated paraoxonase 1 (PON1) activity

HDL-associated paraoxonase 1 (PON1) undergoes inactivation under oxidative stress and is preserved by dietary antioxidants. PON1 cysteines can affect PON1 enzymatic activities. S-Glutathionylation, a redox regulatory mechanism characterized by the formation of a mixed disulfide between a protein thiol and oxidized glutathione (GSSG), was shown to preserve some enzymes from irreversible inactivation under pathological conditions. We questioned whether PON1 activity is regulated by S-glutathionylation. Incubation of PON1 or HDL with GSSG indeed resulted in a dose-dependent inactivation of PON1 activities, including its physiological activity to increase HDL-mediated macrophage cholesterol efflux. This PON1 inactivation was associated with the formation of a mixed disulfide bond between GSSG and PON1's cysteine residue(s), as detected by immunoblotting with anti-glutathione IgG. PON1 activity was recovered following the addition of a reducing agent, DL-Dithiothreitol (DTT), to the PON1-SSG complex. We thus conclude that HDL-associated serum PON1 can undergo S-glutathionylation under oxidative stress with a consequent reversible inactivation.     

Trypanosoma congolense: paraoxonase 1 prolongs survival of infected mice

In vitro studies have suggested that a fraction of human high density lipoprotein (HDL), termed trypanosome lysis factor (TLF), can protect against trypanosome infection. We examined the involvement of two proteins located in the TLF fraction, apolipoprotein A-II (apoA-II) and paraoxonase 1 (PON1), against trypanosome infection. To test whether PON1 is involved in trypanosome resistance, we infected human PON1 transgenic mice, PON1 knockout mice, and wild-type mice with Trypanosoma congolense. When challenged with the same dosage of trypanosomes, mice overexpressing PON1 lived significantly longer than wild-type mice, and mice deficient in PON1 lived significantly shorter. In contrast, mice overexpressing another HDL associated protein, apoA-II, had the same survival as wild-type mice. Together, these data suggest that PON1 provides protection against trypanosome infection. In vitro studies using T. brucei brucei indicated that HDL particles containing PON1 and those depleted of PON1 did not differ in their lysis ability, suggesting that protection by PON1 is indirect. Our data are consistent with an in vivo role of HDL protection against trypanosome infection.     

Hypolipidemic effect of young persimmon fruit in C57BL/6.KOR-ApoEshl mice

We investigated the hypolipidemic effects of young persimmon fruit (YP) on apolipoprotein E-deficient C57BL/6.KOR-ApoEshl mice. These mice exhibited higher plasma cholesterols, except for high-density lipoprotein (HDL), and lower plasma HDL cholesterol than C57BL/6.Cr mice that had the same genetic background as the C57BL/6.KOR-ApoEshl mice. Male C57BL/6.KOR-ApoEshl mice (n=5) were fed a diet supplemented with dry YP, Hachiya-kaki, at a concentration of 5% (w/w) for 10 weeks. YP treatment significantly lowered plasma chylomicron, very low-density lipoprotein (VLDL) and low-density lipoprotein (LDL) cholesterols, and triglyceride, and this response was accompanied by an elevation of fecal bile acid excretion. In the liver, sterol regulatory element binding protein-2 gene expression was significantly higher in mice fed YP, while the mRNA and protein levels of the LDL receptor did not change. These results indicate that acceleration of fecal bile acid excretion is a major mechanism of the hypolipidemic effect induced by YP in C57BL/6.KOR-ApoEshl mice.     

Abnormal fibrillin-1 expression and chronic oxidative stress mediate endothelial mesenchymal transition in a murine model of systemic sclerosis

Systemic sclerosis (SSc) is an autoimmune connective tissue disorder characterized by oxidative stress, impaired vascular function, and attenuated angiogenesis. The tight-skin (Tsk(-/+)) mouse is a model of SSc that displays many of the cellular features of the clinical disease. We tested the hypotheses that abnormal fibrillin-1 expression and chronic phospholipid oxidation occur in Tsk(-/+) mice and, furthermore, that these factors precipitate a prooxidant state, collagen-related protein expression, apoptosis, and mesenchymal transition in endothelial cells cultured on Tsk(-/+) extracellular matrix. Human umbilical vein endothelial cells were seeded on microfibrils isolated from skin of C57BL/6J (control) and Tsk(-/+) mice in the presence or absence of chronic pretreatment with the apolipoprotein Apo A-I mimetic D-4F (1 mg·kg(-1)·day(-1) ip for 6 to 8 wk). Nitric oxide-to-superoxide anion ratio was assessed 12 h after culture, and cell proliferation, apoptosis, and phenotype were studied 72 h after culture. Tsk(-/+) mice demonstrated abnormal "big fibrillin" expression (405 kDa) by Western blot analysis compared with control. Endothelial cells cultured on microfibrils prepared from Tsk(-/+) mice demonstrated reduced proliferation, a prooxidant state (reduced nitric oxide-to-superoxide anion ratio), increased apoptosis, and collagen-related protein expression associated with mesenchymal transition. Chronic D-4F pretreatment of Tsk(-/+) mice attenuated many of these adverse effects. The findings demonstrate that abnormal fibrillin-1 expression and chronic oxidative stress mediate endothelial mesenchymal transition in Tsk(-/+) mice. This mesenchymal transition may contribute to the reduction in angiogenesis that is known to occur in this model of SSc.     

Apolipoprotein E predisposes to obesity and related metabolic dysfunctions in mice

Obesity is a central feature of the metabolic syndrome and is associated with increased risk for insulin resistance and typeII diabetes. Here, we investigated the contribution of human apoliproteinE3 and mouse apoliproteinE to the development of diet-induced obesity in response to western-type diet. Our data show that apolipoproteinE contributes to the development of obesity and other related metabolic disorders, and that human apolipoproteinE3 is more potent than mouse apolipoproteinE in promoting obesity in response to western-type diet. Specifically, we found that apolipoproteinE3 knock-in mice fed western-type diet for 24 weeks became obese and developed hyperglycemia, hyperinsulinemia, hyperleptinemia, glucose intolerance and insulin resistance that were more severe than in C57BL/6 mice. In contrast, apolipoproteinE-deficient mice fed western-type diet for the same period were resistant to diet-induced obesity, had normal plasma glucose, leptin and insulin levels, and exhibited normal responses to glucose tolerance and insulin resistance tests. Furthermore, low-density lipoprotein receptor-deficient mice were more sensitive to the development of diet-induced obesity and insulin resistance than apolipoprotein E-deficient mice, but were still more resistant than C57BL/6 mice, raising the possibility that low-density lipoprotein receptor mediates, at least in part, the effects of apolipoproteinE on obesity. Taken together, our findings suggest that, in addition to other previously identified mechanisms of obesity, apolipoproteinE and possibly the chylomicron pathway are also important contributors to the development of obesity and related metabolic dysfunctions in mice.     

A role for FXR and human FGF-19 in the repression of paraoxonase-1 gene expression by bile acids

Paraoxonase-1 (PON1), an enzyme that metabolizes organophosphate insecticides, is secreted by the liver and transported in the blood complexed to HDL. In humans and mice, low plasma levels of PON1 have also been linked to the development of atherosclerosis. We previously reported that hepatic Pon1 expression was decreased when C57BL/6J mice were fed a high-fat, high-cholesterol diet supplemented with cholic acid (CA). In the current study, we used wild-type and farnesoid X receptor (FXR) null mice to demonstrate that this repression is dependent upon CA and FXR. PON1 mRNA levels were also repressed when HepG2 cells, derived from a human hepatoma, were incubated with natural or highly specific synthetic FXR agonists. In contrast, fibroblast growth factor-19 (FGF-19) mRNA levels were greatly induced by these same FXR agonists. Furthermore, treatment of HepG2 cells with recombinant human FGF-19 significantly decreased PON1 mRNA levels. Finally, deletion studies revealed that the proximal -230 to -96 bp region of the PON1 promoter contains regulatory element(s) necessary for promoter activity and bile acid repression. These data demonstrate that human PON1 expression is repressed by bile acids through the actions of FXR and FGF-19.