PPARs and their metabolic modulation: new mechanisms for transcriptional regulation?


Jorge Plutzky MD, Cardiovascular Division, Harvard Medical School, Brigham and Women’s Hospital, Boston, MA, USA.
(fax: +1 617 525 4366; e-mail: jplutzky@rics.bwh.harvard.edu).


Peroxisome proliferator-activated receptors (PPARs) as ligand-activated nuclear receptors involved in the transcriptional regulation of lipid metabolism, energy balance, inflammation, and atherosclerosis are at the intersection of key pathways involved in the pathogenesis of diabetes and cardiovascular disease. Synthetic PPAR agonists like fibrates (PPAR-α) and thiazolidinediones (PPAR-γ) are in therapeutic use to treat dyslipidaemia and diabetes. Despite strong encouraging in vitro, animal model, and human surrogate marker studies with these agents, recent prospective clinical cardiovascular trials have yielded mixed results, perhaps explained by concomitant drug use, study design, or a lack of efficacy of these agents on cardiovascular disease (independent of their current metabolic indications). The use of PPAR agents has also been limited by untoward effects. An alternative strategy to PPAR therapeutics is better understanding PPAR biology, the nature of natural PPAR agonists, and how these molecules are generated. Such insight might also provide valuable information about pathways that protect against the metabolic problems for which PPAR agents are currently indicated. This approach underscores the important distinction between the effects of synthetic PPAR agonists and the unequivocal biologic role of PPARs as key transcriptional regulators of metabolic and inflammatory pathways relevant to diabetes and atherosclerosis.


The survival of any organism requires efficient energy utilization; this fundamental dependence is mirrored by the need to be able to both store and access energy resources as a function of supply and demand. Humans utilize dietary-derived glucose and long-chain fatty acids as sources of energy. As such, highly regulated systems for storing and mobilizing these natural resources in times of need have evolved over time [1]. These nutritional components provide more than just combustible energy, they also produce a host of diverse cellular signals. In this way, these nutrients provide a mechanism through which the broadly defined outside ‘environment’ can communicate directly to specific cells/tissues in response to either nutrient, energy demand, or nutrient excess. One classic example of nutrient-coupled cellular signalling is the postprandial release of insulin, as evidenced by the multiple distal pathways influenced by insulin signalling. Through this pathway, postprandial responses can be coordinated [2]. These cellular responses to nutrient intake can also be a source of maladaptive pathogenesis, including the relationship between nutrient intake and obesity, diabetes and atherosclerosis, ongoing major issues for Western society.

One distinct and important way in which nutrients like fatty acids direct cellular biology is by inducing specific transcriptional responses in the nucleus [3]. Recent work establishes peroxisome proliferator-activated receptors (PPARs) as one mechanism through which nutrient-driven transcriptional regulation can occur [4–6]. PPARs, ligand-activated transcription factors involved in physiological issues such as energy balance, liquid metabolism, and glucose control, are also released upon PPAR activation therapeutic targets for pathologic conditions like diabetes mellitus and dyslipidaemia [7, 8]. Three different PPAR-γ isotypes have been identified, although the insight into specific roles for each remains limited [55–57]. After briefly introducing PPARs, this review will focus on the connection between specific pathways of lipid metabolism and PPAR activation.

Peroxisome proliferator-activated receptors

Peroxisome proliferator-activated receptors, including the three known isotypes PPAR-α, PPAR-γ, and PPAR-δ, are members of the steroid hormone nuclear receptor superfamily [9, 10]. Like other nuclear receptor family members, PPARs contain both a ligand-binding domain, directing specific interaction with the cognate ligand, and a DNA-binding domain that mediates binding to specific PPAR response elements in the promoter region [11] (Fig. 1). In response to ligand binding, PPARs undergo a conformational change that facilitates the formation of a heterodimeric complex with another ligand-activated nuclear receptor, retinoid X receptor (RXR) [9, 11, 12]. This ligand-induced conformational change also facilitates the binding and release of small accessory molecules that are critical determinants of the transcriptional complex [13, 14]. These accessory molecules include co-repressor proteins, like N-CoR and SMRT, released upon PPAR activation and co-activator proteins, like PPAR-γ co-activator-1(PGC-1), steroid receptor coactivator 1, and CREB-binding protein (Fig. 1) recruited to the activated PPAR [15, 16]. The activity of the PPAR/RXR/accessory molecule transcriptional complex is also determined by phosphorylation. For example, insulin, by inducing PPAR phosphorylation, increases its activity [17]. Thus, many levels of control – ligand, receptor, accessory molecule, target gene promoter – ultimately combine to determine PPAR responses [9]. Moreover, these factors can all also vary in a cell- and tissue-specific manner. Importantly, PPARs can not only induce but also repress gene transcription, the latter occurring perhaps in part through the interaction with other transcription factors such as NF-κB, STAT, AP-1, and NFAT [18]. Together, this complex series of events provides coordinated regulation of a cassette of proteins involved in multiple metabolic processes, including energy homeostasis. Despite these multiple common elements of PPAR activation, each PPAR isotype still maintains a distinct biological role, including their part in lipid metabolism.

Figure 1.

 PPARs – ligand-activated transcription factors. A schematized representation of PPAR biology is shown. PPARs are ligand (L)-activated transcription factors. In response to a specific ligand interacting with the ligand-binding domain, PPAR activation fosters the formation of a heterodimeric transcriptional complex involving the PPAR and the retinoid X receptor (RXR), which has its ligand, purportedly 9 cis retinoic acid. This complex can then interact via DNA-binding domains with PPAR response elements (PPREs in the promoter regions of specific target genes), inducing expression of target genes. PPAR-mediated repression of gene expression can also occur. One important factor determining transcriptional responses is the recruitment or release of small accessory molecules known as co-activators and co-repressors. The three known PPAR isotypes (alpha, gamma, and delta) have many aspects of shared biology although each member of this sub-family also has unique nonoverlapping patterns of tissue expression, ligands, and biological effects [9].

PPAR-α: transcriptional sensor of fatty acids?

PPAR-α was first isolated from mice liver in 1990 by Issemann and Green [19]. In humans, PPAR-α is expressed in liver, heart, kidney, skeletal muscle, intestine, and pancreas, although low levels have been found in many other tissues [20, 21]. Chemical screening led to the observation that fibrates, therapeutic agents used to treat elevated triglycerides and low HDL, are PPAR-α agonists [22]. The more recent observation that PPAR-α is expressed throughout the vasculature and immune cells has suggested direct ways in which PPAR-α activation might be involved in fibrate effects on cardiovascular events as seen in trials such as VA-HIT [23] and the subgroup analysis of the Fenofibrate Intervention and Event Lowering in Diabetes (FIELD) study [24].

PPAR-α activation has been shown to affect transcriptional expression of at least approximately 80–100 genes thus far [23]. These various PPAR-α regulated target genes reveal important roles for PPAR-α in fatty acid oxidation, lipid metabolism, and inflammation [12, 23, 25]. Considerable insight into PPAR-α function has come through the study of the PPAR-α-deficient mouse (PPARα−/−) [26]. The primary defects observed in PPARα−/− mice were impaired hepatic fatty acid uptake and oxidation [27]. Subsequent studies have demonstrated that PPAR-α regulates genes encoding for proteins involved in these processes, namely FATP, acyl-coA oxidase, and cytochrome P450 [28, 29]. These findings are consistent with the observation that this PPAR isoform is increased under conditions of fasting, when increased hepatic fatty acid uptake and fatty acid oxidation is required to manage energy substrates for survival. Of note, during fasting, PPARα−/− mice accumulate lipids in the liver [29]. PPAR-α-deficient mice experience relative hypoglycaemia, as might be expected in their obligate shift towards carbohydrates, i.e. glucose, as an energy resource, given their compromised ability to utilize fatty acids in the absence of PPAR-α [30].

The integral role of PPAR-α in fatty acid oxidation supports a role for this nuclear receptor in the regulation of lipoprotein metabolism. PPAR-α is involved in various central aspects of HDL and triglyceride-rich lipoprotein metabolism, evident through its induction of apolipoprotein A1, a key element in HDL particles; lipoprotein lipase (LPL), a central enzyme in triglyceride metabolism; and repression of apoCIII, the endogenous inhibitor of LPL action [7]. In the absence of PPAR-α, mice manifest impaired HDL-C and triglyceride metabolism and, with age, develop obesity [31, 32]. These various actions of PPAR-α are consistent with the clinical effects of fibrates increasing HDL levels and lowering triglyceride [33].

More recent work has implicated PPAR-α in the regulation of inflammation [3, 34]. In general, these data suggest that PPAR-α activation may limit various inflammatory pathways, for example, inhibiting the production of proinflammatory cytokines such as interleukin-6 as well as repressing cytokine-induced expression of endothelial adhesion molecules such as VCAM1 [35, 36]. PPAR-α activation can also repress induction of tissue factor, a potent procoagulant thought to contribute to plaque thrombogenicity [37]. One common mechanism in PPAR-α-mediated repression of inflammatory targets is inhibition of NF-κΒ [38]. PPAR-α has also been reported to be activated by leukotriene B4, which may help terminate inflammatory responses [39]. Consistent with these reports, PPAR-α-deficient mice manifest basal increases in their inflammatory state, with prolonged responses to pro-inflammatory stimuli [39]. All these results suggest that pathways of lipid metabolism that activate PPAR-α could also exert anti-inflammatory effects [3, 18]. This appears to be the case, as is discussed further below.

Two general lines of evidence suggest these in vitro findings and studies in PPAR-α-deficient mice may extend to humans, at least to some extent. First is the data regarding the effects of lipid-lowering fibrates as reported PPAR-α agonists [10, 22, 33]. As might be predicted for PPAR-α agonists, fibrates like gemfibrozil and fenofibrate lower triglycerides and raise HDL [40]. Fibrates have also been shown to decrease atherosclerosis and lower the incidence of ischaemic cardiovascular events [41, 42]. Limited studies also suggest fibrates can decrease levels of inflammatory markers such as C-reactive protein [43, 44]. Establishing if such effects derive from PPAR-α activation remains difficult. Nevertheless, PPAR-α activation in vitro and in animal models exerts transcriptional responses consistent with the clinical effects seen with fibrates, for example, inducing apoA1, a major component of HDL, and LPL, a central enzyme in triglyceride metabolism [7].

A second line of data for PPAR-α in humans comes from the study of functional PPARα polymorphisms in humans. The substitution of leucine to valine at position 162 results in increased levels of total cholesterol and apoB in patients with Type 2 diabetes, suggesting a link exists between the L162 V allele and diabetic atherogenic dyslipidaemia [43, 44].

It is important to note other studies that suggest possible pathological consequences through PPAR-α activation. For example, PPAR-α has been implicated in hypertension [47, 48]. Elegant studies primarily in mice have also suggested excessive fatty acid metabolism and increased PPAR-α activity in diabetic cardiomyopathy [49]. Although this work is primarily based on overexpression or the absence of PPAR-α in mouse models, the relevance of these findings to humans requires further study. Although there has not been much evidence for hypertension and cardiomyopathy in fibrate trials, the fact remains that currently used fibrates are also not particularly potent PPAR-α agonists. Other subtleties may also be at work such as cell-specific responses and species-specific differences, to name a few. For example, the peroxisome proliferation that occurs in mice with fibrates, and which gave this receptor family its name, is not found in humans [50].

Importantly, these potential adverse effects of synthetic PPAR-α agonists combine with the recent somewhat disappointing results of fenofibrate, a more potent PPAR-α agonist than gemfibrozil, in the FIELD study to underscore a fundamental issue in the PPAR arena: the nature of the endogenous signals that activate PPAR-α remains incompletely defined. The assumption that fibrates faithfully reproduce the effects of natural PPAR-α agonists in vivo, or that fibrates can ‘complement’ defective PPAR-α activation in humans represents a major and potentially flawed assumption. In this regard, it becomes essential to make a distinction between the responses to serendipitously identified synthetic molecules, like PPAR drugs, and the biological role of PPARs themselves. The established position of PPARs at the intersection of energy balance, lipid metabolism, inflammation, and atherosclerosis establishes this issue as a critical one. For PPAR-α, long-chain fatty acids, including polyunsaturated fatty acids such as linoleic acid, linolenic acid, eicosapentaenoic acid (EPA) and arachidonic acid, have been suggested as activators [51–53]. Biological modification of these fatty acids, as with the oxidized forms of EPA, may generate more potent PPAR-α activators [54]. Eicosanoids, including hydroxyl-eicosatetraenoic acid (HETE), 9 and 13 hydroxydecanoic acids (HODEs), and as noted previously, leukotriene B4, have also been reported to be PPAR-α activators [39, 51–53]. Similar issues regarding natural PPAR activators also extend to PPAR-γ.

PPAR-γ: at the cross-roads of adipogenesis, insulin sensitivity, and atherosclerosis?

PPAR-γ is classically characterized by its high expression and functional role in adipose tissue, where it was first identified [58–60]. PPAR-γ is also expressed in skeletal muscle, liver, heart, and intestine [61]. As with PPAR-α, PPAR-γ has also been identified in cells within the vascular and immune systems [62–64].

The serendipitous discovery of synthetic PPAR-γ agonists in the form of the thiazolidinedione drug class provided an invaluable tool for studying this nuclear receptor [9, 65]. Moreover, thiazolidinediones, e.g. pioglitazone and rosiglitazone, have found clinical utility as insulin-sensitizing anti-diabetic agents [66]. As with PPAR-α, the nature of endogenous PPAR-γ ligands has remained more obscure. PPAR-γ has been reported to be activated by long-chain polyunsaturated fatty acids (linoleic, linolenic, and arachidonic acid) [59]. Lysophosphatidic acid has been recently identified as a putative PPAR-γ agonist that could induce neointima formation through a PPAR-γ-dependent mechanism [67, 68]. Other fatty acids including nitrated forms may also act via PPAR-γ [69, 70]. The prostaglandin 15d-prostaglandin J2 (15d-PGJ2) is also reportedly a PPAR-γ agonist, although the functional significance and presence of this molecule remains unclear [65]. 15d-PGJ2 also has PPAR-γ-independent effects as well, for example limiting inflammation through IκB kinase [71]. Certain phospholipids, and the HODEs have also been reported as PPARγ agonists [72, 73].

Extensive data establish PPAR-γ involvement in adipocyte differentiation, lipid storage, and glucose homeostasis, and its transcriptional regulation of a number of genes involved in these processes [74]. For example, PPAR-γ induces expression of the fat-specific adipocyte P2 (aP2) gene, LPL, FATP, FABP, FAT, and ACS, the GLUT4 glucose transporter, c-Cbl-associated protein (CAP), glucokinase, and uncoupling proteins 2 and 3 (UCP2 and UCP 3) [1, 73]. A role for PPAR-γ in limiting inflammation has also been reported, with PPAR-γ ligands inhibiting macrophage activation and the production of inflammatory cytokines such as tumour necrosis factor- α (TNF-α) [76, 77]. Similar effects have been reported in T lymphocytes with evidence that PPAR-γ limits the production of proximal cytokines such as IFN-γ and interleukin-1 (IL-1) [78, 79].

PPAR-γ also plays a role in the balance between lipid influx and efflux in macrophages. Although PPAR-γ activation induces the fatty acid transporter CD36 (FAT) [58], recent work shows that this effect is not associated with foam cell formation [80]. PPAR-γ can also promote lipid efflux by inducing ATP-binding cassette protein A1 (ABCA1) [81, 82]. PPARγ-deficient (−/−) mice have proved difficult to study as they are generally not viable, due to placental dysfunction that leads to impaired cardiac development, although one surviving pup was described with lipodystrophy, fatty liver, and intestinal haemorrhage [81]. PPAR-γ heterozygous deficient (+/−) mice show impaired adipocyte function and glucose homeostasis, and also increased levels of leptin [84]. Interestingly, although synthetic PPAR-γ, PPAR-α agonists decrease glucose levels, mice lacking one PPAR-γ allele have lower glucose levels [85]. One hypothesis offered for this seeming paradox is that these adipocytes are younger, smaller, less differentiated, and hence more sensitive and responsive to insulin and other signals [85]. Conditional knockouts are providing new insights into specific roles for PPAR-γ in other settings, like macrophages [86].

Human PPAR-γ polymorphisms are rare [87]. Polymorphisms that decrease the transcriptional activity of PPAR-γ are associated with insulin resistance, Type 2 diabetes, and hypertension at an early age; substitution of valine to methionine at position 290 and proline to leucine at 467 have been described [88, 89]. Increased transcriptional activity of PPAR-γ, due to the substitution of proline to glutamine at position 115, has been described in three unrelated subjects with obesity [90]. This mutation was found to mediate enhanced adipocyte differentiation. A separate study found no association between this mutation and morbid obesity [91]. A relatively common proline 12 alanine PPAR-γ variant, which may reduce transcriptional activity, has been investigated in population studies and has shown varying associations with body mass index (BMI) and Type 2 diabetes. In a study of middle-aged and elderly Finns, the P12A allele was associated with a lower BMI and improved insulin sensitivity [92]. In support of this finding, studies of diabetic and nondiabetic Japanese and Finnish subjects have reported a lower frequency of the variant in the diabetic group, suggesting that it protects against Type 2 diabetes in these populations [93, 94]. The P12A allele has also been associated with a reduced risk of myocardial infarction [95]. However, in another study, the frequency of the P12A allele did not differ significantly between diabetic subjects and nondiabetic controls [96]. Two independent studies of Caucasian populations in the United States have reported that the variant was associated with a higher BMI and found no association with fasting insulin or glucose levels [97]. Furthermore, two studies reported an association between the variant and Type 2 diabetes (although in one of these studies the association was only observed in women) [98, 99].

PPAR-δ: widely expressed, poorly understood

The ubiquitous expression of PPAR-δ suggests its involvement in basic cellular functions, such as membrane lipid synthesis and turnover [100–102]. It is also believed to play a role in cell proliferation/differentiation [7, 101]. Like PPAR-α, PPAR-δ is activated by long-chain fatty acids, including several polyunsaturated fatty acids and eicosanoids (linoleic acid, arachidonic acid, cPGI, and iloprost) [9]. Erucic acid has been reported to be more specific for PPAR-δ than other PPAR subtypes [103]. Synthetic PPAR-δ agonists have not reached clinical use.

PPAR-δ appears to be the most highly expressed PPAR in skeletal muscle where it reportedly can compensate for PPAR-α deficiency [104]. PPAR-δ has been shown to increase FFA oxidation in muscle, and appears to be involved in the adaptation of muscle to FFA metabolism [7]. In support of this, PPAR-δ activation has been shown to regulate genes involved in fatty acid catabolism (malonyl-CoA decarboxylase, CPT1, and UCP3) [7, 101, 105]. Furthermore, as upregulation of these genes still occurs in the muscle of PPARα−/− mice subjected to exercise or fasting conditions, PPAR-δ may be more relevant to the adaptive response of muscle than PPAR-α [105, 106]. Unfortunately, most PPAR-δ−/− mice die at an early stage; those that do survive demonstrate reduced fat stores, although mice with adipocyte-specific PPAR-δ deletion do not have reduced fat mass [107, 108]. Recent studies do suggest a potential role for PPAR-δ in fat storage. PPAR-δ has also been suggested to mediate lipid accumulation in macrophages in response to very low density lipoprotein (VLDL), possibly by influencing the transcription of adipose differentiation-related protein [109]. This finding raises the question as to how PPAR-δ activation affects atherosclerotic lesion progression. Further studies are needed for clarification, especially as PPAR-δ has also been implicated in limiting inflammation [110].

Lipolytic pathways to PPAR activation

As noted above, multiple lines of evidence in vitro and in vivo reveal PPARs to be closely intertwined with lipid metabolism. Despite this, until recently, very little evidence existed that established how pathways of lipid metabolism might be connected to PPAR responses. Although the prior seminal work establishing fatty acids as potential agonists for PPARs provided critical new insight into these receptors, the data relied on in vitro assays and the direct addition of fatty acids at high concentrations. This information also offered little distinction amongst different fatty acids or explanation for how selective PPAR isoform activation might occur under physiological conditions. The literature clearly establishes that tremendous selectivity and specificity exists amongst pathways of lipid metabolism, with specific lipoprotein particles interacting with multiple different lipases to generate specific lipolytic products. Even amongst fatty acids, biological responses can be widely divergent. Such notions are strongly supported by the nonoverlapping roles that exist for multiple different lipases, each of which has its own unique pattern of expression, substrate selectivity, and biological action.

Our group has pursued a working model that a transcriptional network exists in which specific pathways of lipid metabolism, as defined by different lipoproteins and unique lipases, direct activation of distinct nuclear receptors. As such, we have sought to employ specific pathways of lipid metabolism as a novel way to probe PPAR responses. Several reports from us and others support this concept. The triglyceride lipase family includes LPL, hepatic lipase, and endothelial lipase (EL) [111]. Each of these enzymes has its own relative preference for triglyceride versus phospholipid as well as different patterns of expression and activity. LPL is a major mechanism for the hydrolysis of triglyceride-rich lipoproteins. Synthesized in adipocytes, myocytes, and monocytes/macrophages (MO/Mφ), but not hepatocytes, LPL is transported to the endothelial surface where it binds to heparan sulphate proteoglycans [112]. In this position, dimerized LPL hydrolyzes circulating VLDL (produced by the liver) and chylomicrons (originating from the gut), generating fatty acids for energy through beta oxidation of fatty acids or alternatively for storage and presumed future use. ApoCII is a co-factor for LPL action whereas ApoCIII is an endogenous LPL inhibitor [112]. Interestingly, the promoter for ApoCIII has been shown to be repressed by PPAR-α activation. LPL can also nonenzymatically promote lipid uptake by ‘bridging’ various lipoproteins to cellular receptors like the LDL receptor [113]. Interestingly, the role of LPL in atherosclerosis remains controversial [112]. Bridging may foster pro-atherosclerotic LPL effects, especially in macrophages.

We found that the hydrolysis of VLDL by LPL could robustly activate PPAR-α (Fig. 2). Indeed, probing PPAR activation through LPL action revealed somewhat surprising selectively in terms of the lipoprotein substrate (VLDL>>HDL>LDL) and the PPAR isotype being targeted, with more potent activation seen on PPAR-α than PPAR-δ with very little effect on PPAR-γ (Fig. 2a,b respectively). VLDL hydrolysis by LPL releases molecules, presumably fatty acids, that can displace high-potency synthetic PPAR-α ligands from expressed PPAR-α protein in cell-free assays (Fig. 2c). As such, one can state that LPL action generates actual PPAR-αligands rather than just referring to LPL-mediated PPAR-αactivation. Interestingly, the selectivity seen also extended to different fatty acid-releasing lipases. Although exposing the same human pooled VLDL to either LPL or secretory phospholipased A2 released the same amount of total free fatty acids, the extent of PPAR-α activation diverged completely. The lack of PPAR-α activation seen with PLA2 is potentially consistent with the posited pro-inflammatory effects of this enzyme. Although a prior role for LPL in limiting inflammation has not been purported, we found that LPL-mediated PPAR-α activation was able to replicate the effects of a synthetic PPAR-α agonist, limiting cytokine-induced vascular cell adhesion molecule expression (Fig. 2d). Of note, concurrent work by Ron Evans and colleagues found that VLDL hydrolysis by LPL could also activate PPAR-δ in macrophages [109], suggesting that lipolytic PPAR activation may also vary depending on the cellular context. Together these data offer new ways in which to re-visit the role of LPL in vascular responses and inflammation. For example, the beneficial lipid profile (low triglycerides, increased HDL) and apparent protection from atherosclerosis seen with LPL gain-of-function genetic variants may derive from increased PPAR-α activation [114]. Likewise, the phenotypes seen in association with LPL loss-of-function mutations may be defined by the lack of PPAR agonist generation [115].

Figure 2.

 LPL-treated VLDL generates PPAR-α ligands [117]. (a) LPL treatment of VLDL activates the PPAR-α ligand-binding domain (LBD). Standard PPAR-LBD assays were performed in bovine endothelial cells (EC) co-transfected with PPAR-α- LBD-GAL4 construct, a luciferase PPAR response element (pUASx4-TK-luc), and a β-galactosidase construct for normalization. Transfected cells were then stimulated with pooled human lipoproteins from normal volunteers as shown in either the presence or absence of LPL (30 U mL−1). Details here and below and in Ref. [115]. (b) LPL-treated VLDL (10 μg mL−1) preferentially activates PPAR-α. LBD activation for all three known PPAR isotypes (-α, -γ, -δ) by VLDL in EC was measured in the presence or absence of LPL using standard LBD-GAL4 assays (as in Fig. 1a), and compared to responses induced by known PPAR isotype agonists as follows: PPAR-γ-BRL49653 (also known as rosiglitazone, BRL, 1 μmol L−1); PPAR-α-WY14643 (a research PPARα ligand, WY, 100 μmol L−1), or PPAR-δ activating carbaprostacyclin (carba, 10 μmol L−1). (c) LPL-treated VLDL generates PPAR agonists. LPL-treated VLDL was used in radioligand displacement assays. Competition curves were generated by incubating the LPL/VLDL reaction mixture with specific radiolabelled high potency PPAR ligands (compound A, dual agonist for PPAR-α and PPAR-γ; compound B for just PPAR-δ) and expressed specific PPAR-α, -γ, or -δ protein. 5 nmol L−1 3H2 compound A with GST-hPPAR-α (circles) or GST-hPPAR-γ (triangles), or 2.5 nmol L−1 3H2 compound B with GST-hPPAR-δ (squares). Radioligand displacement in the presence of the indicated concentration of VLDL (0.003–10 μg protein mL−1)/LPL (200 U mL−1 LPL, 37 °C, 1 h) is plotted. (d) Similar to synthetic PPAR-α agonists, LPL-treated VLDL represses cytokine induced VCAM1 in wild-type but not PPAR-α-deficient endothelial cells. VCAM1 surface expression in EC obtained from wild-type (black bars) or PPAR-α−/− mice (white bars) was measured by ELISA using standard protocols. EC cultured in 96-well plates were stimulated with murine TNF-α (10 ng mL−1, 18 h). Data represent absorbance at 410 nm after protein concentration normalization. Both WY14643 (100 μ mol L−1) and LPL/VLDL significantly decreased VCAM1 levels (*, P < 0.005) in wild-type but not PPAR-α-deficient EC. Basal VCAM1 level in PPAR-α−/− is significantly increased when compared with wild-type EC (#, P < 0.001), suggesting PPAR-α plays a basal role in repressing inflammation [117].

These findings expand our view of the nature of lipoprotein and specifically triglyceride metabolism. Although the traditional view holds that the metabolism of triglycerides is a means of handling fatty acids either for their eventual combustion in skeletal muscle and cardiac muscle for energy or their storage in fat for future use and survival, lipoproteins are classically seen as a means of transporting these valuable nutrients (Fig. 3). PPAR activation via lipolysis extends this perspective on triglyceride metabolism and the function of lipoprotein particles, identifying a potential role for lipoproteins in directing transcriptional responses by delivering PPAR agonists. In this context, the action of specific lipases can be understood as a means of accessing those ligands, while dysfunction of lipases can be seen as producing phenotypes that are influenced in part by the absence (or presence) of these activators (Fig. 3). The many known variables and unique attributes of lipoproteins and lipases would thus help define this transcriptional network. Subsequent studies support the idea that such a network exists that is dictated by specific lipase–lipoprotein interactions yielding the activation of distinct nuclear receptors and hence their distal transcriptional effects (Fig. 3).

Figure 3.

 Lipolytic pathways in energy balance and transcriptional regulation. Lipoprotein metabolism is a complex network defined by many factors, including highly variable lipoproteins, like very low density lipoprotein (VLDL), low density lipoproteins (LDL), and high density lipoproteins (HDL) that act as substrates for lipases, a large, loosely defined family of enzymes that can hydrolyze various lipoprotein components. Lipoproteins can be distinguished by their percentage composition of different components (triglycerides, cholesterol, phospholipid, etc.) as well as by modifications such as oxidation, which can generate distinct lipoproteins like mildly oxidized electronegative LDL (LDL−) and more heavily oxidized LDL (LDLox). Lipases also vary in many ways including patterns of expression, preferred substrates, and lipolytic products. Lipoprotein lipase (LPL) and endothelial lipase (EL) are both members of the triacylglycerol lipase family. LPL and EL interaction with these lipoprotein substrates generates a host of lipolytic products including various forms of fatty acids, with these molecules varying in terms of chain length and saturation, as well as other molecules like monoacylglycerol. While fatty acids have traditionally been considered invaluable energy resources, either being combusted for energy or stored for future use in fat, the data discussed here reveal another functional aspect to lipoprotein metabolism: the release of molecules that can direct transcriptional responses. All variables inherent in lipoprotein metabolism at the level of the lipase and lipoprotein can also direct distinct transcriptional responses. Published examples of such lipolytic pathways to PPAR activation include VLDL hydrolysis by LPL [109, 117], hydrolysis of electronegative LDL [118], and HDL hydrolysis by EL [119].

Although lipoprotein substrate preferences for LPL-mediated PPAR activation follows a pattern based on relative triglyceride content (VLDL>>LDL>HDL), the response with LDL was out of proportion to its triglyceride content. Using electronegative LDL as a model substrate that both undergoes LPL hydrolysis and can promote inflammation, we were able to show that LPL can release HODEs, a known PPAR-α agonist, from electronegative LDL, reversing its pro-inflammatory responses. We have also found that HDL hydrolysis by EL can activate PPAR-α, although through a pathway that is distinct from LPL-treated VLDL [109]. These findings offer a novel platform for explaining the anti-inflammatory effects of HDL and also suggest that the presumption that EL exerts solely pro-atherosclerotic effects may not include functional roles EL may play. These issues will be worthy of further exploration including consideration of how other lipases may target non-PPAR fatty acid-activating nuclear receptors.

In considering endogenous nuclear receptor activation, one of the best studied examples is 9 cis retinoic acid (RA) – a purported ligand for RXR. Retinoic acid can be formed from the symmetric cleavage of beta-carotene (Fig. 4). Beta carotene however can also undergo asymmetric cleavage to yield a series of molecules known as apocarotenals (Fig. 4). Although apocarotenals have been suggested to exist in vivo, their functional role remains unknown [116]. We hypothesized that apocarotenals, like 9 cis RA, might also be active modulators of transcription. Indeed, subsequent studies revealed that β-apo-14′ carotenal (apo14) but not other related carotenals could inhibit the activation of PPAR-γ, PPAR-α, and RXR by their respective agonists. Moreover, apo14 had no effect on other multiple nuclear receptors tested. Consistent with these effects, apo14 could repress classic PPAR-α and PPAR-γ responses, including adhesion molecule expression and adipogenesis. Additional studies will be required to understand whether apo14 might play a role helping limit PPAR activation or perhaps explain the untoward effects of β-carotene supplementation in clinical trials.

Figure 4.

 β Apo-14′ carotenal, but not other structurally related apocarotenals, selectively inhibits agonist-induced activation of PPAR-α, PPAR-γ, and RXR. (a) β-carotene can undergo central enzymatic cleavage to generate various retinoids like retinol, retinal, and retinoic acid – molecules that can activate various nuclear receptors such as RAR and RXR. Under certain conditions, β-carotene can also undergo asymmetric cleavage, yield a series of molecules known as apocarotenals (apo14, apo12, apo10, apo8, depending on the length of the side chain). (b) LBD-GAL4 assays (as outlined in Fig. 2) for each of the nuclear receptors shown were performed in bovine EC in the presence of specific ligands for each of these receptors either alone (set as 100%) or after pretreatment with either apo14 or apo8. Whereas apo8 had no effect on any of these responses, apo14 significantly (asterisks, P at least < 0.05) inhibited the response of PPAR-α, -δ, -γ, RXR-α, and LXR-α but none of the other receptors tested. Studies with apo14 on well-established PPAR-α (VCAM1 repression) and PPAR-γ (adipogenesis) responses were also consistent with this repression. For details, see Ref. [120].


Peroxisome proliferator-activated receptors are clearly key regulators of lipid metabolism, energy balance, inflammation, and atherosclerosis. As such, they remain important for better understanding how these pathways are integrated, as well as the nature of dyslipidaemia, diabetes, and cardiovascular disease [10]. A separate issue is the best way to target these intriguing receptors to improve outcomes, independent of the success and disappointment encountered with the currently available agents [121–125]. Greater insight into PPAR biology under endogenous physiological conditions might help in better targeting these receptors for clinical gain while also helping identify mechanisms that protect against those very conditions for which PPAR agonists are prescribed. This insight may also suggest pathways that when defective contribute to pathological conditions through inadequate PPAR ligand generation. Characterization of the exact molecules released from lipoproteins that activate PPARs might offer templates for developing PPAR agonists that may be safer, if not more efficacious.

Conflict of interest statement

No conflict of interest was declared.


We acknowledge the expert assistance of Ruzena Tupy. Editorial support was provided by Drs Emma Kenny and Elinor Washbrook, The Future Forum Secretariat, London, UK. Grant support includes R01 HL071745, R01/RFA HL-075771, P01 HL048743 (all J.P.), and the Donald W. Reynolds Foundation (J.P., W.A.).