Activation of peroxisome proliferator-activated receptor-α enhances fatty acid oxidation in human adipocytes

Peroxisome proliferator-activated receptor-α (PPARα) is a key regulator for maintaining whole-body energy balance. However, the physiological functions of PPARα in adipocytes have been unclarified. We examined the functions of PPARα using human multipotent adipose tissue-derived stem cells as a human adipocyte model. Activation of PPARα by GW7647, a potent PPARα agonist, increased the mRNA expression levels of adipocyte differentiation marker genes such as PPARγ, adipocyte-specific fatty acid-binding protein, and lipoprotein lipase and increased both GPDH activity and insulin-dependent glucose uptake level. The findings indicate that PPARα activation stimulates adipocyte differentiation. However, lipid accumulation was not changed, which is usually observed when PPARγ is activated. On the other hand, PPARα activation by GW7647 treatment induced the mRNA expression of fatty acid oxidation-related genes such as CPT-1B and AOX in a PPARα-dependent manner. Moreover, PPARα activation increased the production of CO₂ and acid soluble metabolites, which are products of fatty acid oxidation, and increased oxygen consumption rate in human adipocytes. The data indicate that activation of PPARα stimulates both adipocyte differentiation and fatty acid oxidation in human adipocytes, suggesting that PPARα agonists could improve insulin resistance without lipid accumulation in adipocytes. The expected effects of PPARα activation are very valuable for managing diabetic conditions accompanied by obesity, because PPARγ agonists, usually used as antidiabetic drugs, induce excessive lipid accumulation in adipocytes in addition to improvement of insulin resistance.     

Peroxisome proliferator-activated receptor-α selective ligand reduces adiposity, improves insulin sensitivity and inhibits atherosclerosis in LDL receptor-deficient mice

Fenofibrate, a selective ¹PPAR-α activator, is prescribed to treat human dyslipidemia. The aim of this study was to delineate the mechanism of fenofibrate-mediated reductions in adiposity, improvements in insulin sensitivity, and lowering of triglycerides (TG) and free fatty acids (FFA) and to investigate if these favorable changes are related to the inhibition of lipid deposition in the aorta. To test this hypothesis we used male LDLr deficient mice that exhibit the clinical features of metabolic syndrome X when fed a high fat high cholesterol (HF) diet. LDLr deficient mice fed HF diet and simultaneously treated with fenofibrate (100 mg/kg body weight) prevented development of obesity, lowered serum triglycerides and cholesterol, improved insulin sensitivity, and prevented accumulation of lipids in the aorta. Lowering of circulating lipids occurred via down-regulation of lipogenic genes, including fatty acid synthase, acetyl CoA carboxylase and diacyl glycerol acyl transferase-2, concomitant with decreased liver TG and cholesterol, and TG output rate. Fenofibrate also suppressed liver apoCIII mRNA levels and markedly increased lipoprotein lipase mRNA levels, known to enhance serum TG catabolism. In addition, fenofibrate profoundly reduced epididymal fat and mesenteric fat mass to the levels seen in lean mice. The reductions in body weight were associated with elevation of hepatic uncoupling protein 2 (UCP2) mRNA, a concomitant increase in the ketone body formation, and improved insulin sensitivity associated with tumor necrosis factor-α reductions and phosphoenol pyruvate carboxykinase down-regulation. These results demonstrate that fenofibrate improves lipid abnormalities partly via inhibition of TG production and partly via clearance of TG-rich apoB particles by elevating LPL and reduced apoCIII. The prevention of obesity development occurred via energy expenditure. Fenofibrate-mediated hypolipidemic effects together with improved insulin sensitivity and loss of adiposity led to the reductions in the aortic lipid deposition by inhibiting early stages of atherosclerosis possibly via vascular cell adhesion molecule-1 (VCAM-1) modulation. These results suggest that potent PPAR-α activators may be useful in the treatment of syndrome X. (Mol Cell Biochem xxx: 1–16, 2005)     

1,25-Dihydroxyvitamin D₃ suppresses inflammation-induced expression of plasminogen activator inhibitor-1 by blocking nuclear factor-κB activation

Plasminogen activator inhibitor (PAI)-1 is a major fibrinolytic inhibitor. High PAI-1 is associated with increased renal and cardiovascular disease risk. Previous studies demonstrated PAI-1 down-regulation by 1,25-dihydroxyvitamin D? (1,25(OH)?D?), but the molecular mechanism remains unknown. Here we show that exposure of mouse embryonic fibroblasts to TNFα or LPS led to a marked induction of PAI-1, which was blunted by 1,25(OH)?D?, NF-κB inhibitor or p65 siRNA, suggesting the involvement of NF-κB in 1,25(OH)?D?-induced repression. In mouse Pai-1 promoter a putative cis-κB element was identified at -299. EMSA and ChIP assays showed that TNF-α increased p50/p65 binding to this κB site, which was disrupted by 1,25(OH)?D?. Luciferase reporter assays showed that PAI-1 promoter activity was induced by TNFα or LPS, and the induction was blocked by 1,25(OH)?D?. Mutation of the κB site blunted TNFα, LPS or 1,25(OH)?D? effects. 1,25(OH)?D? blocked IκBα degradation and arrested p50/p65 nuclear translocation. In mice LPS stimulated PAI-1 expression in the heart and macrophages, and the stimulation was blunted by pre-treatment with a vitamin D analog. Together these data demonstrate that 1,25(OH)?D? down-regulates PAI-1 by blocking NF-κB activation. Inhibition of PAI-1 production may contribute to the reno- and cardio-protective effects of vitamin D.     

The streptokinase–human plasminogen activator complex. Composition and identity of a subcomponent with activator activity

Reactions between purified plasminogen and streptokinase, labelled with ¹³¹I and ¹²⁵I respectively, were investigated by polyacrylamide-gel discontinuous electrophoresis. A molecular complex consisting of both ¹³¹I-labelled plasminogen and ¹²⁵I-labelled streptokinase migrated between plasminogen and streptokinase. This complex contained bovine plasminogen activator activity. The relative quantities of ¹³¹I-labelled plasminogen and ¹²⁵I-labelled streptokinase in this complex were markedly affected by reaction conditions. A fragment that retained 50% or more of the parent activator activity was released from the complex after exposure to mercaptoethanol. This subcomponent had an estimated molecular weight of 70000, and contained both ¹³¹I-labelled plasminogen and ¹²⁵I-labelled streptokinase.     

Forkhead transcription factor FoxO1 inhibits insulin- and transforming growth factor-β-stimulated plasminogen activator inhibitor-1 expression

Elevated levels of plasminogen activator inhibitor-1 (PAI-1) are considered a risk factor for chronic liver disease in patients with hyperinsulinemia. Insulin increases the expression of PAI-1, and inactivates the forkhead box-containing protein FoxO1. We were interested in whether the inactivation of FoxO1 is involved in the activation of PAI-1 expression under conditions of insulin stimulation. Here, we examined whether adenoviral-mediated expression of a constitutively active form of FoxO1 (Ad-CA-FoxO1) inhibited insulin-stimulated PAI-1 expression in human HepG2 hepatocellular liver carcinoma cells and mouse AML12 hepatocytes. Treatment of cells with insulin increased PAI-1 gene expression, and this effect was abolished by Ad-CA-FoxO1. Insulin also increased the transforming growth factor (TGF)-β-induced expression of PAI-1 mRNA, and Ad-CA-FoxO1 inhibited this effect. Transient transfection assays using a reporter gene under the control of the PAI-1 promoter revealed that CA-FoxO1 inhibits Smad3-stimulated PAI-1 promoter activity. Taken together, our results indicate that FoxO1 inhibits PAI-1 expression through the inhibition of TGF-β/Smad-mediated signaling pathways. Our data also suggest that in the hyperinsulinemic state, FoxO1 is inactivated by increased levels of insulin, and does not function as an inhibitor of TGF-β-induced PAI-1 expression.     

Is plasminogen activator inhibitor-1 the molecular switch that governs urokinase receptor-mediated cell adhesion and release?

Induction of the urokinase type plasminogen activator receptor (uPAR) promotes cell adhesion through its interaction with vitronectin (VN) in the extracellular matrix, and facilitates cell migration and invasion by localizing uPA to the cell surface. We provide evidence that this balance between cell adhesion and cell detachment is governed by PA inhibitor-1 (PAI-1). First, we demonstrate that uPAR and PAI-1 bind to the same site in VN (i.e., the amino-terminal somatomedin B domain; SMB), and that PAI-1 competes with uPAR for binding to SMB. Domain swapping and mutagenesis studies indicate that the uPAR-binding sequence is located within the central region of the SMB domain, a region previously shown to contain the PAI-1-binding motif. Second, we show that PAI-1 dissociates bound VN from uPAR and detaches U937 cells from their VN substratum. This PAI-1 mediated release of cells from VN appears to occur independently of its ability to function as a protease inhibitor, and may help to explain why high PAI-1 levels indicate a poor prognosis for many cancers. Finally, we show that uPA can rapidly reverse this effect of PAI-1. Taken together, these results suggest a dynamic regulatory role for PAI-1 and uPA in uPAR-mediated cell adhesion and release.     

Identification,purification, and pharmacological activity analysis of Desmodus rotundus salivary plasminogen activator alpha1 (DSPAα1) expressed in transgenic rabbit mammary glands

Desmodus rotundus plasminogen activator alpha 1(DSPAα1) is a thrombolytic protein with advantages, such as a long half-life, high accuracy and specificity for thrombolysis, wide therapeutic window, and no neurotoxicity. To date, DSPAα1 has only been expressed in the Chinese hamster ovary, insect cells, transgenic tobacco plants, and Pichia pastoris. To the best of our knowledge, we are the first to report the expression of DSPAα1 in transgenic rabbit mammary glands, extract the product, and analyze its pharmacology activity. An efficient mammary gland-specific expression vector pCL25/DSPAα1 was transferred to prokaryotic zygotes in rabbits by microinjection to generate six DSPAα1 transgenic rabbits. The recombinant DSPAα1 (rDSPAα1) expression in transgenic rabbit milk was 1.19?±?0.26 mg/mL. The rDSPAα1 purification protocol included pretreatment, ammonium sulfate precipitation, benzamidine affinity chromatography, cation exchange chromatography, and Cibacron blue affinity chromatography; approximately 98% purity was achieved using gel electrophoresis. According to sequencing results, the primary structure of rDSPAα1 was consistent with the theoretical design sequence, and its molecular weight was consistent with that of the natural protein. N-terminal sequencing results indicated rDSPAα1 to be a mature protein, as the goat signal peptide sequence of the expression vector was no longer detected. The fibrinolytic activity of rDSPAα1 was estimated to be 773,333 IU/mg. Fibrin-agarose plate assay and in vitro rat blood clot degradation assay showed that rDSPAα1 had strong thrombolytic activity. In conclusion, we report recombinant DSPAα1 with high thrombolytic activity expressed in transgenic rabbit mammary glands.

     

Deficiency of lymphotoxin-α does not exacerbate high-fat diet-induced obesity but does enhance inflammation in mice

Lymphotoxin-α (LTα) is secreted by lymphocytes and acts through tumor necrosis factor-α receptors and the LTβ receptor. Our goals were to determine whether LT has a role in obesity and investigate whether LT contributes to the link between obesity and adipose tissue lymphocyte accumulation. LT deficient (LT(-/-)) and wild-type (WT) mice were fed standard pelleted rodent chow or a high-fat/high-sucrose diet (HFHS) for 13 wk. Body weight, body composition, and food intake were measured. Glucose tolerance was assessed. Systemic and adipose tissue inflammatory statuses were evaluated by quantifying plasma adipokine levels and tissue macrophage and T cell-specific gene expression in abdominal fat. LT(-/-) mice were smaller (20%) and leaner (25%) than WT controls after 13 wk of HFHS diet feeding. LT(-/-) mice showed improved glucose tolerance, suggesting that, in WT mice, LT may impair glucose metabolism. Surprisingly, adipose tissue from rodent chow- and HFHS-fed LT(-/-) mice exhibited increased T lymphocyte and macrophage infiltration compared with WT mice. Despite the fact that LT(-/-) mice exhibited an enhanced inflammatory status at the systemic and tissue level even when fed rodent chow, they were protected from enhanced diet-induced obesity and insulin resistance. Thus, LT contributes to body weight and adiposity and is required to modulate the accumulation of immune cells in adipose tissue.     

1,25-Dihydroxyvitamin D3 suppresses inflammation-induced expression of plasminogen activator inhibitor-1 by blocking nuclear factor-κB activation

Plasminogen activator inhibitor (PAI)-1 is a major fibrinolytic inhibitor. High PAI-1 is associated with increased renal and cardiovascular disease risk. Previous studies demonstrated PAI-1 down-regulation by 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃), but the molecular mechanism remains unknown. Here we show that exposure of mouse embryonic fibroblasts to TNFα or LPS led to a marked induction of PAI-1, which was blunted by 1,25(OH)₂D₃, NF-κB inhibitor or p65 siRNA, suggesting the involvement of NF-κB in 1,25(OH)₂D₃-induced repression. In mouse Pai-1 promoter a putative cis-κB element was identified at −299. EMSA and ChIP assays showed that TNF-α increased p50/p65 binding to this κB site, which was disrupted by 1,25(OH)₂D₃. Luciferase reporter assays showed that PAI-1 promoter activity was induced by TNFα or LPS, and the induction was blocked by 1,25(OH)₂D₃. Mutation of the κB site blunted TNFα, LPS or 1,25(OH)₂D₃ effects. 1,25(OH)₂D₃ blocked IκBα degradation and arrested p50/p65 nuclear translocation. In mice LPS stimulated PAI-1 expression in the heart and macrophages, and the stimulation was blunted by pre-treatment with a vitamin D analog. Together these data demonstrate that 1,25(OH)₂D₃ down-regulates PAI-1 by blocking NF-κB activation. Inhibition of PAI-1 production may contribute to the reno- and cardio-protective effects of vitamin D.     

Long-term effects of pubertal stressors on female sexual receptivity and estrogen receptor-α expression in CD-1 female mice

Exposure to stress during puberty can lead to long-term behavioral alterations. Female mice, of the inbred C57BL/6 strain, have been shown to display lower levels of sexual receptivity in adulthood when exposed to shipping stress or to an immune challenge during puberty. The present study investigated whether this effect can be extended to CD1 outbred mice and examined a possible mechanism through which exposure to stressors could suppress sexual receptivity. The results revealed that CD1 mice injected with lipopolysaccharide (LPS) or exposed to shipping stress at 6 weeks old display lower levels of sexual receptivity in response to estradiol and progesterone in adulthood than control mice. Moreover, mice exposed to shipping stress at 8 weeks old also displayed reduced sexual receptivity, but those injected with LPS at that time showed slightly reduced effects, suggesting that the sensitive pubertal period extends to 8 weeks of age in this strain of mice. The examination of estrogen receptor-α (ER-α) expression revealed that mice exposed to shipping stress during the sensitive period (6 weeks) display lower levels of ER-α expression in the medial preoptic area and the ventromedial nucleus and the arcuate nucleus of the hypothalamus than mice shipped at a younger age. These findings support the prediction that exposure to shipping stress or LPS during puberty decreases behavioral responsiveness to estradiol and progesterone in adulthood in an outbred strain of mice through enduring suppression of ER-α expression in some brain areas involved in the regulation of female sexual behavior.     

The pathophysiological function of peroxisome proliferator-activated receptor-γ in lung-related diseases

Research into respiratory diseases has reached a critical stage and the introduction of novel therapies is essential in combating these debilitating conditions. With the discovery of the peroxisome proliferator-activated receptor and its involvement in inflammatory responses of cardiovascular disease and diabetes, attention has turned to lung diseases and whether knowledge of this receptor can be applied to therapy of the human airways. In this article, we explore the prospect of peroxisome proliferator-activated receptor-γ as a marker and treatment focal point of lung diseases such as asthma, chronic obstructive pulmonary disorder, lung cancer and cystic fibrosis. It is anticipated that peroxisome proliferator-activated receptor-γ ligands will provide not only useful mechanistic pathway information but also a possible new wave of therapies for sufferers of chronic respiratory diseases.     

Lycopene attenuates body weight gain through induction of browning via regulation of peroxisome proliferator-activated receptor γ in high-fat diet-induced obese mice

Lycopene (LYC), one of the major carotenoids in tomatoes, has been preclinically and clinically used to obesity and type 2 diabetes management. However, whether its ability of countering body weight gain is related to induction of brown-like adipocyte phenotype in white adipose tissues (WAT) remains largely unknown. Activation of peroxisome proliferator-activated receptor γ (PPARγ) serves the brown-like phenotype conversion and energy expenditure. Here, we show that LYC treatment promotes glucose consumption and improves insulin sensitivity, as well as fosters white adipocytes browning through up-regulating mRNA and protein expression levels of PPARγ, uncoupling protein 1, PPARγ coactivator-1α and PR domain-containing 16 in the differentiated 3T3-L1 adipocytes and primary adipocytes, as well as in the WAT of HFD-exposed obese mice. In addition, LYC treatment attenuates body weight gain and improves serum lipid profiles as well as promotes brown adipose tissue activation in obese mice. Moreover, PPARγ is induced with LYC intervention in mitochondria respiration and browning in white adipocytes and tissues. Taken together, these results suggest that LYC counteracts obesity and improves glucose and lipid metabolism through induction of the browning via up-regulation of PPARγ, which offers a new perspective of this compound to combat obesity and obesity-related disorders.     

Increased peroxisomal fatty acid β-oxidation and enhanced expression of peroxisome proliferator-activated receptor-α in diabetic rat liver

To determine whether the increased fatty acid -oxidation in the peroxisomes of diabetic rat liver is mediated by a common peroxisome proliferation mechanism, we measured the activation of long-chain (LC) and very long chain (VLC) fatty acids catalyzed by palmitoyl CoA ligase (PAL) and lignoceryl CoA ligase and oxidation of LC (palmitic acid) and VLC (lignoceric acid) fatty acids by isotopic methods. Immunoblot analysis of acyl-CoA oxidase (ACO), and Northern blot analysis of peroxisome proliferator-activated receptor (PPAR-), ACO, and PAL were also performed. The PAL activity increased in peroxisomes and mitochondria from the liver of diabetic rats by 2.6-fold and 2.1-fold, respectively. The lignoceroyl-CoA ligase activity increased by 2.6-fold in diabetic peroxisomes. Palmitic acid oxidation increased in the diabetic peroxisomes and mitochondria by 2.5-fold and 2.7-fold, respectively, while lignoceric acid oxidation increased by 2.0-fold in the peroxisomes. Immunoreactive ACO protein increased by 2-fold in the diabetic group. The mRNA levels for PPAR-, ACO and PAL increased 2.9-, 2.8- and 1.6-fold, respectively, in the diabetic group. These results suggest that the increased supply of fatty acids to liver in diabetic state stimulates the expression of PPAR- and its target genes responsible for the metabolism of fatty acids.     

Relationship between lipoprotein lipase and peroxisome proliferator-activated receptor-α expression in rat liver during development

The present study was addressed to determine whether the high expression of peroxisome proliferator-activated receptor-alpha (PPAR-alpha) in rat liver during the perinatal stage plays a role in the induction of liver lipoprotein lipase (LPL) expression and activity. Parallel increases in liver mRNA PPAR-alpha and LPL activity were found in newborn rats, and after a slight decline, values remained elevated until weaning. Anticipated weaning for 3 days caused a decline in those two variables as well as in the mRNA LPL level, and a similar change was also found in liver triacylglycerol concentration. Force-feeding with Intralipid in 10-day-old rats or animals kept fasted for 5 h showed high mRNA-PPARalpha and -LPL levels as well as LPL activity with low plasma insulin and high FFA levels, whereas glucose and a combination of glucose and Intralipid produced low mRNA-PPARalpha and -LPL levels as well as LPL activity. Under these latter conditions, plasma insulin and FFA levels were high in those rats receiving the combination of glucose and Intralipid, whereas plasma FFA levels were low in those force-fed with glucose. It is proposed that the hormonal and nutritional induction of liver PPAR-alpha expression around birth and its maintained elevated level throughout suckling is responsible for the induction of liver LPL-expression and activity during suckling.     

Cardiomyocyte specific peroxisome proliferator-activated receptor-α overexpression leads to irreversible damage in ischemic murine heart

Aims

Peroxisome proliferator-activated receptor (PPAR)-α is downregulated in ischemic myocardium resulting in substrate switch from fatty acid oxidation to glucose utilization. Pharmacological PPAR-α activation leads to increased fatty acid oxidation and myocardial lipotoxicity. The aim of our study was to investigate the role of cardiomyocyte specific PPAR-α overexpression in myocardial adaptation to repetitive ischemic injury without myocardial infarction.

Main methods

Repetitive, brief I/R was performed in male and female MHC-PPAR-α overexpressing and wildtype-C57/Bl6 (WT)-mice, age 10–12 weeks, for 3 and 7 consecutive days. After echocardiography, their hearts were excised for histology and gene/protein-expression measurements (Taqman/Western blot).

Key findings

MHC-PPAR-α mice developed microinfarctions already after 3 days of repetitive I/R in contrast to interstitial fibrosis in WT-mice. We found higher deposition of glycogen, increased apoptosis and dysfunctional regulation of antioxidative mediators in MHC-PPAR-α mice. MHC-PPAR-α mice presented with maladaptation of myosin heavy chain isoforms and worse left ventricular dysfunction than WT-mice. We found prolonged, chemokine-driven macrophage infiltration without induction of proinflammatory cytokines in MHC-PPAR-α mice. Persistent accumulation of myofibroblasts in microinfarctions indicated active remodeling resulting in scar formation in contrast to interstitial fibrosis without microinfarctions in WT-mice. However, MHC-PPAR-α hearts had only a weak induction of tenascin-C in contrast to its strong expression in WT-hearts.

Significance

Cardiomyocyte-specific PPAR-α overexpression led to irreversible cardiomyocyte loss with deteriorated ventricular function during brief, repetitive I/R episodes. We identified higher glycogen deposition, increased apoptosis, deranged antioxidative capacity and maladaptation of contractile elements as major contributors involved in the modulation of post-ischemic inflammation and remodeling.     

Activation of peroxisome proliferator-activated receptor-γ by rosiglitazone improves lipid homeostasis at the adipose tissue-liver axis in ethanol-fed mice

The development of alcohol-induced fatty liver is associated with a reduction of white adipose tissue (WAT). Peroxisome proliferator-activated receptor (PPAR)-γ prominently distributes in the WAT and plays a crucial role in maintaining adiposity. The present study investigated the effects of PPAR-γ activation by rosiglitazone on lipid homeostasis at the adipose tissue-liver axis. Adult C57BL/6 male mice were pair fed liquid diet containing ethanol or isocaloric maltose dextrin for 8 wk with or without rosiglitazone supplementation to ethanol-fed mice for the last 3 wk. Ethanol exposure downregulated adipose PPAR-γ gene and reduced the WAT mass in association with induction of inflammation, which was attenuated by rosiglitazone. Ethanol exposure stimulated lipolysis but reduced fatty acid uptake capacity in association with dysregulation of lipid metabolism genes. Rosiglitazone normalized adipose gene expression and corrected ethanol-induced lipid dyshomeostasis. Ethanol exposure induced steatosis and upregulated inflammatory genes in the liver, which were attenuated by rosiglitazone. Hepatic peroxisomal fatty acid β-oxidation was suppressed by ethanol in associated with inhibition of acyl-coenzyme A oxidase 1. Rosiglitazone elevated plasma adiponectin level and normalized peroxisomal fatty acid β-oxidation rate. However, rosiglitazone did not affect ethanol-reduced very low-density lipoprotein secretion from the liver. These results demonstrated that activation of PPAR-γ by rosiglitazone reverses ethanol-induced adipose dysfunction and lipid dyshomeostasis at the WAT-liver axis, thereby abrogating alcoholic fatty liver.