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1.  Rare loss-of-function mutations in ANGPTL family members contribute to plasma triglyceride levels in humans  
The relative activity of lipoprotein lipase (LPL) in different tissues controls the partitioning of lipoprotein-derived fatty acids between sites of fat storage (adipose tissue) and oxidation (heart and skeletal muscle). Here we used a reverse genetic strategy to test the hypothesis that 4 angiopoietin-like proteins (ANGPTL3, -4, -5, and -6) play key roles in triglyceride (TG) metabolism in humans. We re-sequenced the coding regions of the genes encoding these proteins and identified multiple rare nonsynonymous (NS) sequence variations that were associated with low plasma TG levels but not with other metabolic phenotypes. Functional studies revealed that all mutant alleles of ANGPTL3 and ANGPTL4 that were associated with low plasma TG levels interfered either with the synthesis or secretion of the protein or with the ability of the ANGPTL protein to inhibit LPL. A total of 1% of the Dallas Heart Study population and 4% of those participants with a plasma TG in the lowest quartile had a rare loss-of-function mutation in ANGPTL3, ANGPTL4, or ANGPTL5. Thus, ANGPTL3, ANGPTL4, and ANGPTL5, but not ANGPTL6, play nonredundant roles in TG metabolism, and multiple alleles at these loci cumulatively contribute to variability in plasma TG levels in humans.
doi:10.1172/JCI37118
PMCID: PMC2613476  PMID: 19075393
2.  Angiopoietin-like 4 (Angptl4) 
Adipocyte  2012;1(3):182-187.
Angiopoietin-like 4 (Angptl4) is a secreted protein modulating triacylglycerol homeostasis. Its transcription is induced by glucocorticoids, which act to elevate circulating Angptl4 levels during fasting. In investigating the role of Angptl4 in glucocorticoid action, we identified that in addition to its known ability to inhibit lipoprotein lipase, Angptl4 stimulates intracellular adipocyte lipolysis. Fatty acid release by murine adipocytes following fasting or treatment with glucocorticoids or catecholamines is highly Angptl4-dependent. In fact, Angptl4 can directly stimulate cAMP-dependent PKA signaling and lipolysis when added to adipocytes. Here, we detail this novel Angptl4-dependent lipolytic regulatory mechanism and discuss its physiological and therapeutic implications.
doi:10.4161/adip.20787
PMCID: PMC3609093  PMID: 23700531
Angptl4; adipocyte; cAMP; catecholamine; fasting; glucocorticoids; lipolysis
3.  Lack of Relationship between Cord Serum Angiopoietin-Like Protein 4 (ANGPTL4) and Lipolytic Activity in Human Neonates Born by Spontaneous Delivery 
PLoS ONE  2013;8(12):e81201.
Background
Ligands of peroxisome-proliferator activated receptors (PPARs), such as non-esterified fatty acids (NEFAs), induce expression of angiopoietin-like protein 4 (ANGPTL4). Recently ANGPTL4 has been reported to be a mediator of intracellular adipose lipolysis induced by glucocorticoids.
Objective
To determine the concentrations of ANGPTL4 in cord serum of neonates born by spontaneous vaginal delivery (SVD) and by pre-labor cesarean section (CS) from healthy women, and to relate them to parameters of neonatal lipolytic activity at birth.
Measurements
In 54 neonates born by SVD and in 56 neonates born by CS, arterial cord blood was drawn to determine insulin, cortisol, triacylglycerols (TAGs), glycerol, non-esterified fatty acids (NEFAs), individual fatty acids, ANGPTL4, adiponectin, retinol binding protein 4 (RBP4) and leptin.
Results
Birth weight and neonatal fat mass in SVD and CS showed no difference, but the concentrations of glycerol, adiponectin, RBP4, NEFAs and most individual fatty acids were higher in cord serum of neonates born by SVD compared to CS, indicating a higher adipose tissue breakdown in the SVD group. The concentrations of TAG and cortisol were also higher and that of insulin was lower in cord serum of SVD compared to the CS group. However, the concentration in cord serum of ANGPTL4 did not differ between the two groups and no positive correlation with either NEFA or glycerol concentrations were detected.
Conclusion
ANGPTL4 is known to stimulate lipolysis in adults, but does not appear to mediate the increased activity in SVD, indicating the presence of different regulatory inputs.
doi:10.1371/journal.pone.0081201
PMCID: PMC3852178  PMID: 24324678
4.  Angptl4 serves as an endogenous inhibitor of intestinal lipid digestion☆ 
Molecular Metabolism  2013;3(2):135-144.
Dietary triglycerides are hydrolyzed in the small intestine principally by pancreatic lipase. Following uptake by enterocytes and secretion as chylomicrons, dietary lipids are cleared from the bloodstream via lipoprotein lipase. Whereas lipoprotein lipase is inhibited by several proteins including Angiopoietin-like 4 (Angptl4), no endogenous regulator of pancreatic lipase has yet been identified. Here we present evidence that Angptl4 is an endogenous inhibitor of dietary lipid digestion. Angptl4−/− mice were heavier compared to their wild-type counterparts without any difference in food intake, energy expenditure or locomotor activity. However, Angptl4−/− mice showed decreased lipid content in the stools and increased accumulation of dietary triglycerides in the small intestine, which coincided with elevated luminal lipase activity in Angptl4−/− mice. Furthermore, recombinant Angptl4 reduced the activity of pancreatic lipase as well as the lipase activity in human ileostomy output. In conclusion, our data suggest that Angptl4 is an endogenous inhibitor of intestinal lipase activity.
doi:10.1016/j.molmet.2013.11.004
PMCID: PMC3953698  PMID: 24634819
Angptl4; Pancreatic lipase; Intestine; Obesity
5.  Linking nutritional regulation of Angptl4, Gpihbp1, and Lmf1 to lipoprotein lipase activity in rodent adipose tissue 
BMC Physiology  2012;12:13.
Background
Lipoprotein lipase (LPL) hydrolyzes triglycerides in lipoproteins and makes fatty acids available for tissue metabolism. The activity of the enzyme is modulated in a tissue specific manner by interaction with other proteins. We have studied how feeding/fasting and some related perturbations affect the expression, in rat adipose tissue, of three such proteins, LMF1, an ER protein necessary for folding of LPL into its active dimeric form, the endogenous LPL inhibitor ANGPTL4, and GPIHBP1, that transfers LPL across the endothelium.
Results
The system underwent moderate circadian oscillations, for LPL in phase with food intake, for ANGPTL4 and GPIHBP1 in the opposite direction. Studies with cycloheximide showed that whereas LPL protein turns over rapidly, ANGPTL4 protein turns over more slowly. Studies with the transcription blocker Actinomycin D showed that transcripts for ANGPTL4 and GPIHBP1, but not LMF1 or LPL, turn over rapidly. When food was withdrawn the expression of ANGPTL4 and GPIHBP1 increased rapidly, and LPL activity decreased. On re-feeding and after injection of insulin the expression of ANGPTL4 and GPIHBP1 decreased rapidly, and LPL activity increased. In ANGPTL4−/− mice adipose tissue LPL activity did not show these responses. In old, obese rats that showed signs of insulin resistance, the responses of ANGPTL4 and GPIHBP1 mRNA and of LPL activity were severely blunted (at 26 weeks of age) or almost abolished (at 52 weeks of age).
Conclusions
This study demonstrates directly that ANGPTL4 is necessary for rapid modulation of LPL activity in adipose tissue. ANGPTL4 message levels responded very rapidly to changes in the nutritional state. LPL activity always changed in the opposite direction. This did not happen in Angptl4−/− mice. GPIHBP1 message levels also changed rapidly and in the same direction as ANGPTL4, i.e. increased on fasting when LPL activity decreased. This was unexpected because GPIHBP1 is known to stabilize LPL. The plasticity of the LPL system is severely blunted or completely lost in insulin resistant rats.
doi:10.1186/1472-6793-12-13
PMCID: PMC3562520  PMID: 23176178
Gene expression; Insulin; Gene inactivation; Cycloheximide; Actinomycin D; Transcription; Translation; Posttranslational
6.  Genetic Variation in ANGPTL4 Provides Insights into Protein Processing and Function*S⃞ 
The Journal of Biological Chemistry  2009;284(19):13213-13222.
Angiopoietin-like protein 4 (ANGPTL4) is a secreted protein that modulates the disposition of circulating triglycerides (TG) by inhibiting lipoprotein lipase (LPL). Here we examine the steps involved in the synthesis and post-translational processing of ANGPTL4, and the effects of a naturally occurring sequence variant (E40K) that is associated with lower plasma TG levels in humans. Expression of the wild-type and mutant proteins in HEK-293A cells indicated that ANGPTL4 formed dimers and tetramers in cells prior to secretion and cleavage of the protein. After cleavage at a canonical proprotein convertase cleavage site (161RRKR164), the oligomeric structure of the N-terminal domain was retained whereas the C-terminal fibrinogen-like domain dissociated into monomers. Inhibition of cleavage did not interfere with oligomerization of ANGPTL4 or with its ability to inhibit LPL, whereas mutations that prevented oligomerization severely compromised the capacity of the protein to inhibit LPL. ANGPTL4 containing the E40K substitution was synthesized and processed normally, but no monomers or oligomers of the N-terminal fragments accumulated in the medium; medium from these cells failed to inhibit LPL activity. Parallel experiments performed in mice recapitulated these results. Our findings indicate that oligomerization, but not cleavage, of ANGPTL4 is required for LPL inhibition, and that the E40K substitution destabilizes the protein after secretion, preventing the extracellular accumulation of oligomers and abolishing the ability of the protein to inhibit LPL activity.
doi:10.1074/jbc.M900553200
PMCID: PMC2676053  PMID: 19270337
7.  ANGPTL4 variants E40K and T266M are associated with lower fasting triglyceride levels in Non-Hispanic White Americans from the Look AHEAD Clinical Trial 
BMC Medical Genetics  2011;12:89.
Background
Elevated triglyceride levels are a risk factor for cardiovascular disease. Angiopoietin-like protein 4 (Angptl4) is a metabolic factor that raises plasma triglyceride levels by inhibiting lipoprotein lipase (LPL). In non-diabetic individuals, the ANGPTL4 coding variant E40K has been associated with lower plasma triglyceride levels while the T266M variant has been associated with more modest effects on triglyceride metabolism. The objective of this study was to determine whether ANGPTL4 E40K and T266M are associated with triglyceride levels in the setting of obesity and T2D, and whether modification of triglyceride levels by these genetic variants is altered by a lifestyle intervention designed to treat T2D.
Methods
The association of ANGPTL4 E40K and T266M with fasting triglyceride levels was investigated in 2,601 participants from the Look AHEAD Clinical Trial, all of whom had T2D and were at least overweight. Further, we tested for an interaction between genotype and treatment effects on triglyceride levels.
Results
Among non-Hispanic White Look AHEAD participants, ANGPTL4 K40 carriers had mean triglyceride levels of 1.61 ± 0.62 mmol/L, 0.33 mmol/L lower than E40 homozygotes (p = 0.001). Individuals homozygous for the minor M266 allele (MAF 30%) had triglyceride levels of 1.75 ± 0.58 mmol/L, 0.24 mmol/L lower than T266 homozygotes (p = 0.002). The association of the M266 with triglycerides remained significant even after removing K40 carriers from the analysis (p = 0.002). There was no interaction between the weight loss intervention and genotype on triglyceride levels.
Conclusions
This is the first study to demonstrate that the ANGPTL4 E40K and T266M variants are associated with lower triglyceride levels in the setting of T2D. In addition, our findings demonstrate that ANGPTL4 genotype status does not alter triglyceride response to a lifestyle intervention in the Look AHEAD study.
doi:10.1186/1471-2350-12-89
PMCID: PMC3146919  PMID: 21714923
8.  Short-Chain Fatty Acids Stimulate Angiopoietin-Like 4 Synthesis in Human Colon Adenocarcinoma Cells by Activating Peroxisome Proliferator-Activated Receptor γ 
Molecular and Cellular Biology  2013;33(7):1303-1316.
Angiopoietin-like protein 4 (ANGPTL4/FIAF) has been proposed as a circulating mediator between the gut microbiota and fat storage. Here, we show that transcription and secretion of ANGPTL4 in human T84 and HT29 colon adenocarcinoma cells is highly induced by physiological concentrations of short-chain fatty acids (SCFA). SCFA induce ANGPTL4 by activating the nuclear receptor peroxisome proliferator activated receptor γ (PPARγ), as demonstrated using PPARγ antagonist, PPARγ knockdown, and transactivation assays, which show activation of PPARγ but not PPARα and PPARδ by SCFA. At concentrations required for PPARγ activation and ANGPTL4 induction in colon adenocarcinoma cells, SCFA do not stimulate PPARγ in mouse 3T3-L1 and human SGBS adipocytes, suggesting that SCFA act as selective PPARγ modulators (SPPARM), which is supported by coactivator peptide recruitment assay and structural modeling. Consistent with the notion that fermentation leads to PPAR activation in vivo, feeding mice a diet rich in inulin induced PPAR target genes and pathways in the colon. We conclude that (i) SCFA potently stimulate ANGPTL4 synthesis in human colon adenocarcinoma cells and (ii) SCFA transactivate and bind to PPARγ. Our data point to activation of PPARs as a novel mechanism of gene regulation by SCFA in the colon, in addition to other mechanisms of action of SCFA.
doi:10.1128/MCB.00858-12
PMCID: PMC3624264  PMID: 23339868
9.  Stable over-expression of PPARβ/δ and PPARγ to examine receptor signaling in human HaCaT keratinocytes 
Cellular signalling  2011;23(12):2039-2050.
Peroxisome proliferator-activated receptor-β/δ (PPARβ/δ) function and receptor cross-talk with other nuclear receptors, including PPARγ and retinoic acid receptors (RARs), was examined using stable human HaCaT keratinocyte cell lines over-expressing PPARβ/δ or PPARγ. Enhanced ligand-induced expression of two known PPAR target genes, adipocyte differentiation-related protein (ADRP) and angiopoietin-like protein 4 (ANGPTL4), was found in HaCaT keratinocytes over-expressing PPARβ/δ or PPARγ. Over-expression of PPARβ/δ did not modulate the effect of a PPARγ agonist on up-regulation of ADRP or ANGPTL4 mRNA in HaCaT keratinocytes. All-trans retinoic acid (atRA) increased expression of a known RAR target gene, yet despite a high ratio of fatty acid binding protein 5 (FABP5) to cellular retinoic acid binding protein II, did not increase expression of ANGPTL4 or 3-phosphoinositide-dependent-protein kinase 1 (PDPK1), even in HaCaT keratinocytes expressing markedly higher levels of PPARβ/δ. While PPARβ/δ-dependent attenuation of staurosporine- or UVB-induced poly (ADP-ribose) polymerase (PARP) cleavage was not observed, PPARβ/δ- and PPARγ-dependent repression of UVB-induced expression and secretion of inflammatory cytokines was found in HaCaT keratinocytes over-expressing PPARβ/δ or PPARγ. These studies suggest that FABP5 does not transport atRA or GW0742 to PPARβ/δ and promote anti-apoptotic activity by increasing expression of PDPK1, or that PPARβ/δ interferes with PPARγ transcriptional activity. However, these studies demonstrate that stable over-expression of PPARβ/δ or PPARγ significantly increases the efficacy of ligand activation and represses UVB-induced expression of tumor necrosis factor α (TNFα), interleukin 6 (IL6), or IL8 in HaCaT keratinocytes, thereby establishing an excellent model to study the functional role of these receptors in human keratinocytes.
doi:10.1016/j.cellsig.2011.07.020
PMCID: PMC3184348  PMID: 21843636
human keratinocytes; PPARβ/δ; PPARγ; retinoic acid; apoptosis; inflammation
10.  Angiopoietin-Like 4 Regulates Epidermal Differentiation 
PLoS ONE  2011;6(9):e25377.
The nuclear hormone receptor PPARβ/δ is integral to efficient wound re-epithelialization and implicated in epidermal maturation. However, the mechanism underlying the latter process of epidermal differentiation remains unclear. We showed that ligand-activated PPARβ/δ indirectly stimulated keratinocyte differentiation, requiring de novo gene transcription and protein translation. Using organotypic skin cultures constructed from PPARβ/δ- and angiopoietin-like 4 (ANGPTL4)-knockdown human keratinocytes, we showed that the expression of ANGPTL4, a PPARβ/δ target gene, is essential for the receptor mediated epidermal differentiation. The pro-differentiation effect of PPARβ/δ agonist GW501516 was also abolished when keratinocytes were co-treated with PPARβ/δ antagonist GSK0660 and similarly in organotypic skin culture incubated with blocking ANGPTL4 monoclonal antibody targeted against the C-terminal fibrinogen-like domain. Our focused real-time PCR gene expression analysis comparing the skin biopsies from wildtype and ANGPTL4-knockout mice confirmed a consistent down-regulation of numerous genes involved in epidermal differentiation and proliferation in the ANGPTL4-knockout skin. We further showed that the deficiency of ANGPTL4 in human keratinocytes and mice skin have diminished expression of various protein kinase C isotypes and phosphorylated transcriptional factor activator protein-1, which are well-established for their roles in keratinocyte differentiation. Chromatin immunoprecipitation confirmed that ANGPTL4 stimulated the activation and binding of JUNB and c-JUN to the promoter region of human involucrin and transglutaminase type 1 genes, respectively. Taken together, we showed that PPARβ/δ regulates epidermal maturation via ANGPTL4-mediated signalling pathway.
doi:10.1371/journal.pone.0025377
PMCID: PMC3178651  PMID: 21966511
11.  Down-regulation of lipoprotein lipase increases glucose uptake in L6 muscle cells 
Thiazolidinediones (TZDs) are synthetic hypoglycemic agents used to treat type 2 diabetes. TZDs target the peroxisome proliferator activated receptor-gamma (PPAR-γ) and improve systemic insulin sensitivity. The contributions of specific tissues to TZD action, or the downstream effects of PPAR-γ activation, are not very clear. We have used a rat skeletal muscle cell line (L6 cells) to demonstrate that TZDs directly target PPAR-γ in muscle cells. TZD treatment resulted in a significant repression of lipoprotein lipase (LPL) expression in L6 cells. This repression correlated with an increase in glucose uptake. Down-regulation of LPL message and protein levels using siRNA resulted in a similar increase in insulin-dependent glucose uptake. Thus, LPL down-regulation improved insulin sensitivity independent of TZDs. This finding provides a novel method for the management of insulin resistance.
doi:10.1016/j.bbrc.2009.08.081
PMCID: PMC2749911  PMID: 19698699
PPAR-γ; Ciglitazone; lipoprotein lipase; skeletal muscle; rat L6 cells; glucose uptake; siRNA
12.  Ligand activation of peroxisome proliferator-activated receptor-β/δ (PPAR β/δ) inhibits cell growth in a mouse mammary gland cancer cell line 
Cancer letters  2009;288(2):219.
The effects of ligand activation of PPARβ/δ were examined in the mouse mammary tumor cell line (C20). Expression of PPARβ/δ was markedly lower in C20 cells as compared to the human non-tumorigenic mammary gland derived cell line (MCF10A) and mouse keratinocytes. Ligand activation of PPARβ/δ in C20 cells caused upregulation of the PPARβ/δ target gene angiopoietin-like 4 (Angptl4). Inhibition of C20 cell proliferation and clonogenicity was observed following treatment with GW0742 or GW501516, two highly specific PPARβ/δ ligands. In addition, an increase in apoptosis was observed in C20 cells cultured with 10 µM GW501516 that preceded the observed inhibition of cell proliferation. Results from this study show that proliferation of the C20 mouse mammary gland cancer cell line is inhibited by ligand activation of PPARβ/δ due in part to increased apoptosis.
doi:10.1016/j.canlet.2009.07.006
PMCID: PMC2815252  PMID: 19660859
peroxisome proliferator-activated receptor-β/δ; mammary gland cancer; cell proliferation; apoptosis
13.  Exome Sequencing, ANGPTL3 Mutations, and Familial Combined Hypolipidemia 
The New England journal of medicine  2010;363(23):2220-2227.
SUMMARY
We sequenced all protein-coding regions of the genome (the “exome”) in two family members with combined hypolipidemia, marked by extremely low plasma levels of low-density lipoprotein (LDL) cholesterol, high-density lipoprotein (HDL) cholesterol, and triglycerides. These two participants were compound heterozygotes for two distinct nonsense mutations in ANGPTL3 (encoding the angiopoietin-like 3 protein). ANGPTL3 has been reported to inhibit lipoprotein lipase and endothelial lipase, thereby increasing plasma triglyceride and HDL cholesterol levels in rodents. Our finding of ANGPTL3 mutations highlights a role for the gene in LDL cholesterol metabolism in humans and shows the usefulness of exome sequencing for identification of novel genetic causes of inherited disorders. (Funded by the National Human Genome Research Institute and others.)
doi:10.1056/NEJMoa1002926
PMCID: PMC3008575  PMID: 20942659
14.  Peroxisome Proliferator-Activated Receptor-β/δ Regulates Angiogenic Cell Behaviors and Oxygen-Induced Retinopathy 
Purpose.
To develop new therapies against ocular neovascularization (NV), we tested the effect of peroxisome proliferator-activated receptor-β/δ (PPAR-β/δ) agonism and antagonism on angiogenic behaviors and in human retinal microvascular endothelial cells (HRMEC) and on preretinal NV in rat oxygen-induced retinopathy (OIR).
Methods.
HRMECs were treated with the PPAR-β/δ agonist GW0742 and the antagonist GSK0660. Messenger RNA levels of a PPAR-β/δ target gene, angiopoietin-like-4 (angptl4) were assayed by qRT-PCR. HRMEC proliferation and tube formation were assayed according to standard protocols. OIR was induced in newborn rats by exposing them to alternating 24-hour episodes of 50% and 10% oxygen for 14 days. OIR rats were treated with GW0742 or GSK0660. Angptl4 protein levels were assessed by ELISA and preretinal NV was quantified by adenosine diphosphatase staining.
Results.
GW0742 significantly increased angptl4 mRNA, and GSK0660 significantly decreased angptl4 mRNA. GW0742 had no effect on HRMEC proliferation, but caused a significant and dose-responsive increase in tube formation. GSK0660 significantly reduced serum-induced HRMEC proliferation and tube formation in a dose-dependent manner. Intravitreal injection of GW0742 significantly increased total retinal Angptl4 protein, but intravitreal injection of GSK0660 had no effect. Intravitreal injection of GW0742 significantly increased retinal NV, as did GW0742 administered by oral gavage. Conversely, both intravitreal injection and intraperitoneal injection of GSK0660 significantly reduced retinal NV.
Conclusions.
PPAR-β/δ activation exacerbates, and its inhibition reduces, preretinal NV. PPAR-β/δ may regulate preretinal NV through a prodifferentiation/maturation mechanism that depends on Angptl4. Pharmacologic inhibition of PPAR-β/δ may provide a rational basis for therapeutic targeting of ocular NV.
The goal of this study was to determine the effect of pharmacologic manipulation of PPAR-β/δ on retinal angiogenesis. We found that inhibition of PPAR-β/δ reduces neovascularization in the rat model of oxygen-induced retinopathy.
doi:10.1167/iovs.13-11608
PMCID: PMC3687964  PMID: 23716627
retinopathy of prematurity; nuclear transcription factor; angiogenesis; vascular endothelial growth factor; peroxisome proliferator-activated receptor
15.  Balanced pan-PPAR activator bezafibrate in combination with statin: comprehensive lipids control and diabetes prevention? 
All fibrates are peroxisome proliferators-activated receptors (PPARs)-alpha agonists with ability to decrease triglyceride and increase high density lipoprotein- cholesterol (HDL-C). However, bezafibrate has a unique characteristic profile of action since it activates all three PPAR subtypes (alpha, gamma and delta) at comparable doses. Therefore, bezafibrate operates as a pan-agonist for all three PPAR isoforms. Selective PPAR gamma agonists (thiazolidinediones) are used to treat type 2 diabetes mellitus (T2DM). They improve insulin sensitivity by up-regulating adipogenesis, decreasing free fatty acid levels, and reversing insulin resistance. However, selective PPAR gamma agonists also cause water retention, weight gain, peripheral edema, and congestive heart failure. The expression of PPAR beta/ delta in essentially all cell types and tissues (ubiquitous presence) suggests its potential fundamental role in cellular biology. PPAR beta/ delta effects correlated with enhancement of fatty acid oxidation, energy consumption and adaptive thermogenesis. Together, these data implicate PPAR beta/delta in fuel combustion and suggest that pan-PPAR agonists that include a component of PPAR beta/delta activation might offset some of the weight gain issues seen with selective PPAR gamma agonists, as was demonstrated by bezafibrate studies. Suggestively, on the whole body level all PPARs acting as one orchestra and balanced pan-PPAR activation seems as an especially attractive pharmacological goal. Conceptually, combined PPAR gamma and alpha action can target simultaneously insulin resistance and atherogenic dyslipidemia, whereas PPAR beta/delta properties may prevent the development of overweight. Bezafibrate, as all fibrates, significantly reduced plasma triglycerides and increased HDL-C level (but considerably stronger than other major fibrates). Bezafibrate significantly decreased prevalence of small, dense low density lipoproteins particles, remnants, induced atherosclerotic plaque regression in thoracic and abdominal aorta and improved endothelial function. In addition, bezafibrate has important fibrinogen-related properties and anti-inflammatory effects. In clinical trials bezafibrate was highly effective for cardiovascular risk reduction in patients with metabolic syndrome and atherogenic dyslipidemia. The principal differences between bezafibrate and other fibrates are related to effects on glucose level and insulin resistance. Bezafibrate decreases blood glucose level, HbA1C, insulin resistance and reduces the incidence of T2DM compared to placebo or other fibrates. Currently statins are the cornerstone of the treatment and prevention of cardiovascular diseases related to atherosclerosis. However, despite the increasing use of statins as monotherapy for low density lipoprotein- cholesterol (LDL-C) reduction, a significant residual cardiovascular risk is still presented in patients with atherogenic dyslipidemia and insulin resistance, which is typical for T2DM and metabolic syndrome. Recently, concerns were raised regarding the development of diabetes in statin-treated patients. Combined bezafibrate/statin therapy is more effective in achieving a comprehensive lipid control and residual cardiovascular risk reduction. Based on the beneficial effects of pan-PPAR agonist bezafibrate on glucose metabolism and prevention of new-onset diabetes, one could expect a neutralization of the adverse pro-diabetic effect of statins using the strategy of a combined statin/fibrate therapy.
doi:10.1186/1475-2840-11-140
PMCID: PMC3502168  PMID: 23150952
Atherogenic dyslipidemia; Bezafibrate; Combined fibrate/statin therapy; Metabolic syndrome; PPAR; Prevention; Residual cardiovascular risk; Type 2 diabetes
16.  Oxidative metabolism of linoleic acid modulates PPAR-beta/delta suppression of PPAR-gamma activity 
Oncogene  2006;25(8):1225-1241.
Peroxisome proliferator–activated receptors (PPARs) are transcription factors that strongly influence molecular events in normal and cancer cells. PPAR-beta/delta overexpression suppresses the activity of PPAR-gamma and -alpha. This interaction has been questioned, however, by studies with synthetic ligands of PPARs in PPAR-beta/delta–null cells, and it is not known whether an interaction between PPAR-beta/delta and -gamma exists, especially in relation to the signaling by natural PPAR ligands. Oxidative metabolites of linoleic and arachidonic acids are natural ligands of PPARs. 13-S-hydroxyoctadecadienoic acid (13-S-HODE), the main product of 15-lipoxygenase-1 (15-LOX-1) metabolism of linoleic acid, downregulates PPAR-beta/delta. We tested (a) whether PPAR-beta/delta expression modulates PPAR-gamma activity in experimental models of the loss and gain of PPAR-beta/delta function in colon cancer cells and (b) whether 15-LOX-1 formation of 13-S-HODE influences the interaction between PPAR-beta/delta and PPAR-gamma. We found that (a) 15-LOX-1 formation of 13-S-HODE promoted PPAR-gamma activity, (b) PPAR-beta/delta expression suppressed PPAR-gamma activity in models of both loss and gain of PPAR-beta/delta function, (c) 15-LOX-1 activated PPAR-gamma by downregulating PPAR-beta/delta , and (d) 15-LOX-1 expression induced apoptosis in colon cancer cells via modulating PPAR-beta/delta suppression of PPAR-gamma. These findings elucidate a novel mechanism of the signaling by natural ligands of PPARs, which involves modulating the interaction between PPAR-beta/delta and PPAR-gamma.
doi:10.1038/sj.onc.1209160
PMCID: PMC1432095  PMID: 16288226
17.  Activating Effect of Benzbromarone, a Uricosuric Drug, on Peroxisome Proliferator-Activated Receptors 
PPAR Research  2007;2007:36092.
Benzbromarone, a uricosuric drug, reportedly causes hepatic hypertrophy accompanied by proliferation of peroxisomes in rats. To elucidate the mechanisms underlying induction of peroxisome proliferation by benzbromarone, we examined binding affinity for peroxisome proliferator-activated receptor α (PPARα) and γ (PPARγ), and effects on the binding activity of PPARs with peroxisome proliferation-responsive element (PPRE) and expression of the PPARs target protein. Binding affinity of benzbromarone for PPARα and PPARγ was examined by reporter gene assay. Binding activity of PPARs with PPRE was determined by electric mobility shift assay, and expression of lipoprotein lipase (LPL) and acyl-CoA synthetase (ACS) by Western blot method. Benzbromarone displayed affinity for PPARα and PPARγ, and promoted binding of PPARs to PPRE. Furthermore, cultured cells with benzbromarone added showed upregulated expression of LPL and ACS. These results suggest that benzbromarone induces peroxisome proliferation in hepatocytes by binding to PPARs, and controls expression of proteins related to lipid metabolism.
doi:10.1155/2007/36092
PMCID: PMC2233808  PMID: 18274627
18.  Intronic Cis-Regulatory Modules Mediate Tissue-Specific and Microbial Control of angptl4/fiaf Transcription 
PLoS Genetics  2012;8(3):e1002585.
The intestinal microbiota enhances dietary energy harvest leading to increased fat storage in adipose tissues. This effect is caused in part by the microbial suppression of intestinal epithelial expression of a circulating inhibitor of lipoprotein lipase called Angiopoietin-like 4 (Angptl4/Fiaf). To define the cis-regulatory mechanisms underlying intestine-specific and microbial control of Angptl4 transcription, we utilized the zebrafish system in which host regulatory DNA can be rapidly analyzed in a live, transparent, and gnotobiotic vertebrate. We found that zebrafish angptl4 is transcribed in multiple tissues including the liver, pancreatic islet, and intestinal epithelium, which is similar to its mammalian homologs. Zebrafish angptl4 is also specifically suppressed in the intestinal epithelium upon colonization with a microbiota. In vivo transgenic reporter assays identified discrete tissue-specific regulatory modules within angptl4 intron 3 sufficient to drive expression in the liver, pancreatic islet β-cells, or intestinal enterocytes. Comparative sequence analyses and heterologous functional assays of angptl4 intron 3 sequences from 12 teleost fish species revealed differential evolution of the islet and intestinal regulatory modules. High-resolution functional mapping and site-directed mutagenesis defined the minimal set of regulatory sequences required for intestinal activity. Strikingly, the microbiota suppressed the transcriptional activity of the intestine-specific regulatory module similar to the endogenous angptl4 gene. These results suggest that the microbiota might regulate host intestinal Angptl4 protein expression and peripheral fat storage by suppressing the activity of an intestine-specific transcriptional enhancer. This study provides a useful paradigm for understanding how microbial signals interact with tissue-specific regulatory networks to control the activity and evolution of host gene transcription.
Author Summary
Recent studies have revealed that the community of microorganisms residing in the intestine regulates fat storage. Microbes evoke this response in part by suppressing expression of the Angptl4 gene, which encodes a secreted inhibitor of fat storage. Although Angptl4 is expressed in multiple tissues, microbial suppression occurs only in the intestine. To determine how microbes control fat storage, we must elucidate the mechanisms underlying intestine-specific and microbial regulation of Angptl4 expression. Here, we take advantage of the unique features of the zebrafish model to define the regulatory DNA sequences controlling angptl4 expression. Our results reveal that different DNA regulatory regions within the angptl4 gene mediate expression of angptl4 in the intestine and other tissues. By assessing the evolution of angptl4 regulatory regions and subjecting them to structure-function analyses, we identify discrete DNA sequences that are required for intestinal expression. Strikingly, microbes suppress the activity of the intestine-specific regulatory region similar to the endogenous angptl4 gene. Therefore, intestinal microbes might regulate angptl4 production by suppressing the signaling pathway interpreted by an intestine-specific transcriptional regulatory region. Our results provide new mechanistic insights into how intestinal microbes might influence fat storage and contribute to the development of obesity.
doi:10.1371/journal.pgen.1002585
PMCID: PMC3315460  PMID: 22479192
19.  Dual and pan-peroxisome proliferator-activated receptors (PPAR) co-agonism: the bezafibrate lessons 
There are three peroxisome proliferator-activated receptors (PPARs) subtypes which are commonly designated PPAR alpha, PPAR gamma and PPAR beta/delta. PPAR alpha activation increases high density lipoprotein (HDL) cholesterol synthesis, stimulates "reverse" cholesterol transport and reduces triglycerides. PPAR gamma activation results in insulin sensitization and antidiabetic action. Until recently, the biological role of PPAR beta/delta remained unclear. However, treatment of obese animals by specific PPAR delta agonists results in normalization of metabolic parameters and reduction of adiposity. Combined treatments with PPAR gamma and alpha agonists may potentially improve insulin resistance and alleviate atherogenic dyslipidemia, whereas PPAR delta properties may prevent the development of overweight which typically accompanies "pure" PPAR gamma ligands. The new generation of dual-action PPARs – the glitazars, which target PPAR-gamma and PPAR-alpha (like muraglitazar and tesaglitazar) are on deck in late-stage clinical trials and may be effective in reducing cardiovascular risk, but their long-term clinical effects are still unknown. A number of glitazars have presented problems at a late stage of clinical trials because of serious side-effects (including ragaglitazar and farglitazar). The old and well known lipid-lowering fibric acid derivative bezafibrate is the first clinically tested pan – (alpha, beta/delta, gamma) PPAR activator. It is the only pan-PPAR activator with more than a quarter of a century of therapeutic experience with a good safety profile. Therefore, bezafibrate could be considered (indeed, as a "post hoc" understanding) as an "archetype" of a clinically tested pan-PPAR ligand. Bezafibrate leads to considerable raising of HDL cholesterol and reduces triglycerides, improves insulin sensitivity and reduces blood glucose level, significantly lowering the incidence of cardiovascular events and new diabetes in patients with features of metabolic syndrome. Clinical evidences obtained from bezafibrate-based studies strongly support the concept of pan-PPAR therapeutic approach to conditions which comprise the metabolic syndrome. However, from a biochemical point of view, bezafibrate is a PPAR ligand with a relatively low potency. More powerful new compounds with pan-PPAR activity and proven long-term safety should be highly effective in a clinical setting of patients with coexisting relevant lipid and glucose metabolism disorders.
doi:10.1186/1475-2840-4-14
PMCID: PMC1236941  PMID: 16168052
20.  Extracellular matrix-bound angiopoietin-like 4 inhibits endothelial cell adhesion, migration, and sprouting and alters actin cytoskeleton 
Circulation Research  2006;99(11):1207-1215.
Angiopoietin-like 4 (ANGPTL4) is a secreted protein which belongs to the angiopoietin family and is involved in angiogenesis and metabolism regulation. We previously reported the induction of angptl4 by hypoxia in endothelial cells and in human ischemic tissues from peripheral artery disease. We here observed in a mouse model of hindlimb ischemia that the mRNA upregulation in the vessels correlates with the accumulation of the full-length protein in ischemic tissues. We then investigated its functions in endothelial cells. In response to hypoxia, endogenous ANGPTL4 accumulates in the subendothelial extracellular matrix (ECM). Whereas the secreted protein undergoes proteolysis leading to truncated fragments present in the medium, only full-length ANGPTL4 interacts with the ECM. Competition and direct binding assays indicate that the strong interaction of ANGPTL4 with the ECM is heparin/heparan sulfate proteoglycans dependent. The balance between matrix-associated and soluble forms of ANGPTL4 points out the role of the ECM in the regulation of its bioavailability. The angiogenic function of the ECM-bound full-length protein was investigated using either the form associated with the conditioned ECM from ANGPTL4-transfected HEK293 cells, or the purified immobilized protein. We show that matrix-associated as well as immobilized ANGPTL4 limit the formation of actin stress fibers and focal contacts in the adhering endothelial cells and inhibits their adhesion. Immobilized ANGPTL4 also decreases motility of endothelial cells and inhibits the sprouting and tube formation. Altogether, these findings show that hypoxic endothelial cells accumulate ANGPTL4 in the ECM, which in turn negatively regulates their angiogenic capacities through an autocrine pathway.
doi:10.1161/01.RES.0000250758.63358.91
PMCID: PMC2241731  PMID: 17068295
Actins; metabolism; Animals; Anoxia; metabolism; physiopathology; Biological Availability; Blood Proteins; metabolism; pharmacology; Cell Adhesion; drug effects; Cell Movement; drug effects; Cells; Cultured; Cytoskeleton; drug effects; ultrastructure; Endothelial Cells; metabolism; Extracellular Matrix; metabolism; Heparin; analogs & derivatives; metabolism; Hindlimb; blood supply; Ischemia; metabolism; Mice; Mice; Inbred C57BL; Neovascularization; Physiologic; drug effects; Proteoglycans; metabolism; Angionenesis; Hypoxia
21.  Ligand-activated PPARβ efficiently represses the induction of LXR-dependent promoter activity through competition with RXR 
Angiopoietin-like protein 3 (angptl3), a member of the vascular endothelial growth factor family, was shown to play an important role in regulating lipid metabolism. To elucidate the mechanism by which PPARβ represses angptl3 promoter activity, reporter constructs were prepared and transfection analysis carried out. PPARβ repressed angptl3-Luc promoter activity and activation of PPARβ by L-165041, a PPARβ-specific ligand, increased the extent of repression. The repression by L-165041 was lost in angptl3-Luc plasmids having a deleted or mutated LXRα binding site (DR4). PPARβL405R, deficient in RXRα binding, had no effect on angptl3-Luc promoter activity. PPARβ did not repress the activity of GAL4-LXRα which activates of GAL4DBD TK-Luc independent of RXR. Addition of RXRα completely abolished the repression of angptl3-Luc activity by PPARβ. Mammalian two-hybrid analysis revealed that PPARβ ligand binding enhanced the dissociation of the LXRα-RXRα heterodimer. Gel shift assays also indicated that PPARβ ligand binding increased dissociation of LXRα/RXRα binding to a DR4 oligonucleotide probe; addition of RXRα restored the binding lost by addition of PPARβ. Collectively, these results suggest that the binding of PPARβ-specific ligand enhances the affinity between RXRα and activated PPARβ and thus may regulate angptl3 gene expression through a DR4 element by competing with LXRα for RXRα.
doi:10.1016/j.mce.2006.05.005
PMCID: PMC1544360  PMID: 16806672
PPAR; PPARβ/δ; Triglyceride; Angiopoietin; LXR; RXR; angptl3, mouse angiopoietin-like 3; LXR, liver X receptor; RXR, retinoid X receptor; PPAR, peroxisome proliferator-activated receptor; FXR, farnesoid-X-receptor; VLDL, very low density lipoprotein; HF, high fat
22.  Muscle-Derived Angiopoietin-Like Protein 4 Is Induced by Fatty Acids via Peroxisome Proliferator–Activated Receptor (PPAR)-δ and Is of Metabolic Relevance in Humans 
Diabetes  2009;58(3):579-589.
OBJECTIVE— Long-chain fatty acids (LCFAs) contribute to metabolic homeostasis in part via gene regulation. This study's objective was to identify novel LCFA target genes in human skeletal muscle cells (myotubes).
RESEARCH DESIGN AND METHODS— In vitro methods included culture and treatment of human myotubes and C2C12 cells, gene array analysis, real-time RT-PCR, Western blotting, ELISA, chromatin immunoprecipitation, and RNA interference. Human subjects (two cohorts) were characterized by oral glucose tolerance test, hyperinsulinemic-euglycemic clamp, magnetic resonance imaging and spectroscopy, and standard blood analyses (glucose, insulin, C-peptide, and plasma lipids).
RESULTS— We show here that ANGPTL4 (encoding angiopoietin-like protein 4) represents a prominent LCFA-responsive gene in human myotubes. LCFA activated peroxisome proliferator-activated receptor (PPAR)-δ, but not PPAR-α or -γ, and pharmacological activation of PPAR-δ markedly induced ANGPTL4 production and secretion. In C2C12 myocytes, knockdown of PPARD, but not of PPARG, blocked LCFA-mediated ANGPTL4 induction, and LCFA treatment resulted in PPAR-δ recruitment to the ANGPTL4 gene. In addition, pharmacological PPAR-δ activation induced LIPE (encoding hormone-sensitive lipase), and this response crucially depended on ANGPTL4, as revealed by ANGPTL4 knockdown. In a human cohort of 108 thoroughly phenotyped subjects, plasma ANGPTL4 positively correlated with fasting nonesterified fatty acids (P = 0.0036) and adipose tissue lipolysis (P = 0.0012). Moreover, in 38 myotube donors, plasma ANGPTL4 levels and adipose tissue lipolysis in vivo were reflected by basal myotube ANGPTL4 expression in vitro (P = 0.02, both).
CONCLUSIONS— ANGPTL4 is produced by human myotubes in response to LCFA via PPAR-δ, and muscle-derived ANGPTL4 seems to be of systemic relevance in humans.
doi:10.2337/db07-1438
PMCID: PMC2646056  PMID: 19074989
23.  Hepatic Proprotein Convertases Modulate HDL Metabolism 
Cell metabolism  2007;6(2):129-136.
SUMMARY
The risk of atherosclerosis is inversely associated with plasma levels of high-density lipoprotein cholesterol (HDL-C). However, HDL metabolism is incompletely understood, and there are few effective approaches to modulate HDL-C levels. Here we show that inhibition in the liver of the classical proprotein convertases (PCs), but not the atypical PCs S1P and PCSK9, decreases plasma HDL-C levels. This metabolic effect of hepatic PCs is critically dependent on expression of endothelial lipase (EL), an enzyme that directly hydrolyzes HDL phospholipids and promotes its catabolism. Hepatic PCs reduce EL function through direct inactivating cleavage of EL as well as through activating cleavage of angiopoietin-like protein 3 (ANGPTL3), an endogenous inhibitor of EL. Thus, inhibition of hepatic PCs results in increased EL activity, leading to reduced HDL-C as well as impaired reverse cholesterol transport. The hepatic PC-ANGPTL3-EL-HDL pathway is therefore a novel mechanism controlling HDL metabolism and cholesterol homeostasis.
doi:10.1016/j.cmet.2007.07.009
PMCID: PMC2565575  PMID: 17681148
24.  Activation of Peroxisome Proliferator–Activated Receptor-β/-δ (PPAR-β/-δ) Ameliorates Insulin Signaling and Reduces SOCS3 Levels by Inhibiting STAT3 in Interleukin-6–Stimulated Adipocytes 
Diabetes  2011;60(7):1990-1999.
OBJECTIVE
It has been suggested that interleukin (IL)-6 is one of the mediators linking obesity-derived chronic inflammation with insulin resistance through activation of STAT3, with subsequent upregulation of suppressor of cytokine signaling 3 (SOCS3). We evaluated whether peroxisome proliferator–activated receptor (PPAR)-β/-δ prevented activation of the IL-6-STAT3-SOCS3 pathway and insulin resistance in adipocytes.
RESEARCH DESIGN AND METHODS
Adipocytes and white adipose tissue from wild-type and PPAR-β/-δ-null mice were used to evaluate the effect of PPAR-β/-δ on the IL-6-STAT3-SOCS3 pathway.
RESULTS
First, we observed that the PPAR-β/-δ agonist GW501516 prevented both IL-6–dependent reduction in insulin-stimulated Akt phosphorylation and glucose uptake in adipocytes. In addition, this drug treatment abolished IL-6–induced SOCS3 expression in differentiated 3T3-L1 adipocytes. This effect was associated with the capacity of the drug to prevent IL-6–induced STAT3 phosphorylation on Tyr705 and Ser727 residues in vitro and in vivo. Moreover, GW501516 prevented IL-6–dependent induction of extracellular signal–related kinase (ERK)1/2, a serine-threonine-protein kinase involved in serine STAT3 phosphorylation. Furthermore, in white adipose tissue from PPAR-β/-δ–null mice, STAT3 phosphorylation (Tyr705 and Ser727), STAT3 DNA-binding activity, and SOCS3 protein levels were higher than in wild-type mice. Several steps in STAT3 activation require its association with heat shock protein 90 (Hsp90), which was prevented by GW501516 as revealed in immunoprecipitation studies. Consistent with this finding, the STAT3-Hsp90 association was enhanced in white adipose tissue from PPAR-β/-δ–null mice compared with wild-type mice.
CONCLUSIONS
Collectively, our findings indicate that PPAR-β/-δ activation prevents IL-6–induced STAT3 activation by inhibiting ERK1/2 and preventing the STAT3-Hsp90 association, an effect that may contribute to the prevention of cytokine-induced insulin resistance in adipocytes.
doi:10.2337/db10-0704
PMCID: PMC3121427  PMID: 21617181
25.  ATGL-mediated fat catabolism regulates cardiac mitochondrial function via PPAR-α and PGC-1 
Nature medicine  2011;17(9):1076-1085.
Peroxisome proliferator-activated receptors (PPARs) are nuclear hormone receptors that regulate genes involved in energy metabolism and inflammation. For biological activity, PPARs require cognate lipid ligands, heterodimerization with retinoic × receptors, and coactivation by PPAR-γ coactivator-1α or PPAR-γ coactivator-1β (PGC-1α or PGC-1β, encoded by Ppargc1a and Ppargc1b, respectively). Here we show that lipolysis of cellular triglycerides by adipose triglyceride lipase (patatin-like phospholipase domain containing protein 2, encoded by Pnpla2; hereafter referred to as Atgl) generates essential mediator(s) involved in the generation of lipid ligands for PPAR activation. Atgl deficiency in mice decreases mRNA levels of PPAR-α and PPAR-δ target genes. In the heart, this leads to decreased PGC-1α and PGC-1β expression and severely disrupted mitochondrial substrate oxidation and respiration; this is followed by excessive lipid accumulation, cardiac insufficiency and lethal cardiomyopathy. Reconstituting normal PPAR target gene expression by pharmacological treatment of Atgl-deficient mice with PPAR-α agonists completely reverses the mitochondrial defects, restores normal heart function and prevents premature death. These findings reveal a potential treatment for the excessive cardiac lipid accumulation and often-lethal cardiomyopathy in people with neutral lipid storage disease, a disease marked by reduced or absent ATGL activity.
doi:10.1038/nm.2439
PMCID: PMC3244833  PMID: 21857651

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