PPAR-γ: A nuclear receptor with affinity for cannabinoids

Life Sciences 77 (2005) 1674 – 1684 www.elsevier.com/locate/lifescie

PPAR-g: A nuclear receptor with affinity for cannabinoids Sumner BursteinT Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, 364 Plantation St., Worcester, MA 01605-2324, USA

Abstract An increasing number of cannabinoid actions are being reported that do not appear to be mediated by either CB1 or CB2, the known cannabinoid receptors. One such example is the synthetic analog ajulemic acid (AJA), which shows potent analgesic and anti-inflammatory effects in rodents and humans. AJA binds weakly to CB1 only at concentrations many fold higher than its therapeutic range, and is, therefore, completely free of psychotropic effects in both normal subjects and pain patients suggesting the involvement of a target site other than CB1. AJA as well as several other cannabinoids appear to have profound effects on cellular lipid metabolism as evidenced by their ability to transform fibroblasts into adipocytes where the accumulation of lipid droplets can be readily observed. Such transformations can be mediated by the activation of the nuclear receptor PPAR-g. A variety of small molecule ligands including AJA have been shown to induce the activation of PPAR-g and, in some cases this has led to the introduction of clinically useful agents. It is suggested that PPAR-g may serve a receptor function for certain actions of some cannabinoids. D 2005 Elsevier Inc. All rights reserved. Keywords: Ajulemic acid; Cannabinoid; Receptor; PPAR-g

The nuclear receptor PPAR-; Peroxisome proliferator-activated receptors (PPARs) are members of the nuclear receptor superfamily that have an important role in regulation of lipid metabolism, hepatic peroxisomal enzyme expression,

T Tel.: +1 508 856 2850; fax: +1 508 856 2003. E-mail address: [email protected]. 0024-3205/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2005.05.039

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insulin sensitivity and glucose homeostasis (Celi and Shuldiner, 2002) PPARs have been classified into three subtypes encoded by different genes: PPARa, PPARy, and PPARg (Shearer and Hoekstra, 2003). Each subtype appears to be differently expressed in a tissue-specific manner because of their binding to specific consensus DNA sequences, known as peroxisome proliferator response elements (PPREs). PPARs act as heterodimers with a retinoid X receptor and, upon binding an agonist, interact with specific cofactors resulting in an increase in the rate of transcription initiation. Potent synthetic PPAR ligands, including the fibrates and thiazolidinediones, have proven effective in the treatment of dyslipidemia and diabetes. The use of such ligands has allowed researchers to elucidate many potential roles for the PPARs in pathological states including atherosclerosis, inflammation, cancer, infertility, and demyelination. For recent reviews, see (Gregoire, 2001; Moller, 2001; Chawla et al., 2001). A partial list of the genes regulated in vivo by PPAR-g is shown in Table 1 (Berger and Moller, 2002). Fig. 1 depicts the molecular features of PPAR-g role as a mediator of the inflammatory response. As indicated, eicosanoids and fatty acids are endogenous ligands for PPAR-g, prostaglandins of the J series and polyunsaturated fatty acids have been studied in this role (Fig. 2). In the category of drugs, antidiabetic drugs, certain NSAIDs and the synthetic cannabinoid ajulemic acid (Liu et al., 2003) appear to manifest some of their actions by activation of PPAR-g (Fig. 3).

The known cannabinoid (CB) receptors At one time it was thought that cannabinoids acted through non-receptor mediated processes such as membrane perturbation. This belief was largely due to the highly lipophyllic properties of Table 1 Genes regulated in vivo by PPAR-g agonists (Adapted from Berger and Moller, 2002) Gene

Regulation

Potential function(s)

aP2 adipocyte fatty acid binding protein Acyl-CoA synthetase PEPCKphosphoenolpyruvate carboxykinase LPL-lipoprotein lipase CD36 FATP-1 Uncoupling protein 1UCP1 UCP3 (+/UCP2) Carnitine palmitoyl transferase1 CPT1 c-CBL-associated protein Insulin receptor substrate-2IRS-2 Pyruvate dehydrogenase kinase 4PDK4

WAT WAT WAT WAT WAT WAT muscle BAT WAT WAT WAT WAT WAT WAT muscle

Adipocyte complement-related factor 30Acrp30

WAT

TNF-a

WAT

Leptin 11h-Hydroxysteroid dehydrogenase 11h-HSD-1

WAT WAT (liver)

Intracellular fatty acid binding Lipogenesis and/or catabolism Glycerol synthesis (for triglycerides) Hydrolysis of triglyceride-containing particles Cell surface fatty acid transporter Cell surface fatty acid transporter Uncouple mitochondrial respiration Uncouple mitochondrial respiration Translocation of fatty acids into mitochondria Insulin signaling toward glucose transport Insulin receptor-mediated signaling Inhibition of pyruvate dehydrogenase(inhibition of glucose oxidation) Fat-specific secreted protein; beneficial metabolic effects on liver/muscle (?) Pro-inflammatory cytokine; potential mediator of insulin resistance Fat-derived hormone that inhibits food intake Controls intracellular conversion to active cortisol

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S. Burstein / Life Sciences 77 (2005) 1674–1684 Ajulemic acid, eicosanoids, fatty acids & NSAIDs

9-cis-retinoic acid

Nuclear membrane

RXR

PPAR-γγ

AJA

DBD

AF-1

Transcription

LBD

H

AF-2

Anti-inflammatory activity

Cytokine expression

Fig. 1. PPAR-g: structure and function. AF-1, ligand-independent activation domain; AF-2, ligand-dependent activation domain; AJA, ajulemic acid; DBD, DNA binding domain; H, hinge region; LBD, ligand binding domain; RXR, retinoic acid receptor. H N

O

O

O

S

N Et • HCl

PIOGLITAZONE HCL (ACTOS) (Rx for Type 2 diabetes)

HO2C

(CH2)3

Z

Z

Z

Z

(CH2)4 Me

ARACHIDONIC ACID (weak endogenous ligand) (CH 2) 3

Z

CO2H

S E

E

(CH 2) 4 Me

O

15-DEOXY-∆12,∆14-PROSTAGLANDIN J2 (potent endogenous ligand)

Cl

HO2C

CH2

NH Cl Diclofenac (NSAID)

Fig. 2. Examples of PPAR-g ligands. Certain fatty acids and prostaglandins as well as NSAIDs and anti-diabetic agents such as the TZDs can activate PPAR-g.

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A

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AJA

Gal4 DBD UAS

TK

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B 1200 1000 800 600 400 200 0 VEHICLE

GW34 1µM

AJA 20 µM

Normalized Luciferase Activity

C 1600 1400 1200 1000 800 600 400 200 0 0

10

20

30

40

50

60

AJA (µM) Fig. 3. AJA activates PPAR-g in a heterologous reporter system (Liu et al., 2003). (A) A schematic of the GAL4 based reporter system. Briefly, PPAR-g was expressed as a Gal4 DBD fusion protein, which will bind to the UAS promoter containing 4 copies of the GAL4 binding sites upstream of the minimal TK promoter. Binding of PPAR-g ligands, such as AJA, will activate the luciferase reporter gene expression. (B) PPAR-g was significantly activated by 20 AM of AJA or 1 AM of GW347845, a potent activator of PPAR-g that served as a positive control. (C) A dose-dependent activation of PPAR-g by AJA. The estimated EC-50 is about 13 AM in this assay.

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cannabinoids and the unsuccessful attempts to identify high affinity binding sites. The situation was changed about 10 years ago with the serendipitous discovery and cloning of two membrane bound receptors (for reviews, see Di Marzo et al., 2002; Pertwee and Ross, 2002). The first, called CB1, is expressed primarily in the CNS, while the second called CB2 is found mainly in the immune system and they have significant homology to each other. Both are seven transmembrane G-protein coupled receptors (GPCR) that are negatively coupled to adenylate cyclase and positively coupled to MAP kinase through Gi/o. CB1 is also believed to mediate arachidonic acid release and can close 5-HT3 receptor ion channels. Functional endpoints due to CB1 activation include impaired cognition and memory and altered motor function as well as CB-induced analgesia. Less is known about the functional consequences of CB2 activation. The characterization of these receptors has led to the discovery of an endogenous cannabinoid system that includes ligands such as anandamide and 2-arachidonylglycerol (2-AG) as well as several synthetic antagonists.

Biological actions of cannabinoids not mediated by CB1 or CB2 A number of cannabinoid actions do not seem to be mediated by either CB1 or CB2 (Pertwee, 1999, 2001; Pertwee and Ross, 2002). To illustrate, although anandamide can act through CB1 and CB2 receptors, it is also a vanilloid receptor (VR1) agonist and it may possess other important modes of action. For example, it is a potent vasodilator of isolated vascular preparations, however, its mechanism of action is uncertain. The cannabinoid CB1 receptor antagonist SR141716A did not inhibit the vasodilator effect of anandamide. Other endogenous (2-arachidonylglycerol, palmitylethanolamide) and synthetic (WIN 55,212-2, CP 55,940) CB1 and CB2 receptor agonists could not mimic this action of anandamide. The selective vanilloid receptor antagonist capsazepine inhibited anandamide-induced vasodilation. In patch-clamp experiments on cells expressing the cloned vanilloid receptor (VR1), anandamide induced a capsazepine-sensitive current in whole cells and isolated membrane patches. Thus, the vanilloid receptor may be another molecular target for the endocannabinoid anandamide, in addition to cannabinoid receptors, in the nervous and cardiovascular systems. (Zygmunt et al., 1999). Other examples are the cannabinoid acids that show many of the actions of CB1/CB2 agonists, however, they do not have significant affinity for either of these receptors (Burstein, 1999). The synthetic analog, AJA, likewise does not bind to either CB1 or CB2 and has recently been shown in a human trial to be an effective analgesic and to be free of psychotropic activity (Karst et al., 2003). The mechanism of action for AJA is currently being studied and may involve mediation by cytokines and/or eicosanoids (Zurier et al., 2003).

Cannabinoids and PPAR-;: ajulemic acid, NAGly and 2-AG Very recent findings suggest a possible candidate for the ajulemic acid receptor, namely, PPAR-g (Liu et al., 2003). The data shown in Fig. 3 illustrate AJA activation of this nuclear receptor that in turn could regulate cellular processes such as the expression of cytokines, lipid metabolism and glucose homeostasis. A PPAR-g specific ligand GW347845 was used as a positive control (Cobb et

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al., 1998). AJA also shows a moderate binding affinity for PPAR-g (Liu et al., 2003), which has been shown to have a large hydrophobic ligand binding domain that may serve as the binding site. In addition, the endogenous ligands for PPAR-g include several eicosanoids suggesting a possible indirect activation mechanism for AJA since it is known that AJA, like many other cannabinoids, stimulates the arachidonic acid cascade resulting in increased prostaglandin synthesis (Burstein, unpublished data). Another example is the activation of PPAR-g by the endocannabinoid, 2-AG, that has recently been the subject of a preliminary report (Rockwell and Kaminski, 2003). They showed that this endogenous cannabinoid receptor agonist induced differentiation of murine fibroblasts (3T3-L1) into adipocytes as evidenced by the use of the Oil Red assay (vide infra). This, as well as other data, suggested that 2-AG is an endogenous ligand for PPAR-g. An increase in the incorporation of [14C]-oleic acid into triglycerides (TGs) with tetrahydrocannabinol (THC) as well as AJA has been reported (Recht et al., 2001). C6 rat glioma cells were incubated with ajulemic acid for 24 and 48 h and the effects measured by thin layer radio chromatographic methods. This provides an independent corroboration for the Oil Red assay data that measure primarily cellular triglyceride levels. It also further supports the possibility that a number of cannabinoids may have the ability to activate PPAR-g. Studies on possible PPAR-g activation using the endogenous analog of anandamide, Narachidonylglycine (NAGly) (Huang et al., 2001) have been carried out (Burstein and Karim, unpublished data). Data obtained using the Oil Red assay (vide infra) are shown in Fig. 4. In this experiment a mouse macrophage cell line (RAW) was treated with the endogenous cannabinoid NAGly. The assay was performed as described below and the results shown in Fig. 4 panel A indicate a dose-related increase in stainable lipid droplets. To account for changes in total cell number, an identical experiment was done and assayed using the methylthiazoletetrazolium (MTT) assay. Fig. 4 panel B shows the result when the two assays are expressed as a ratio of values. These data suggest that PPAR-g in RAW cells is activated by NAGly resulting in a profound change in lipid metabolism that is characteristic of differentiation to adipocyte-like cells. These preliminary findings further suggest that the cannabinoid-induced effect is more general than the original observation with ajulemic acid (Liu et al., 2003).

The Oil Red assay as an indicator of PPAR-;activation Oil Red O is a highly lipophyllic dye substance that was discovered in 1969 and has been used extensively in histology to visualize areas rich in neutral lipids such as triglycerides and cholesteryl esters. It produces an intense red color due to its light absorption characteristics; k max 518 nm. PPAR-g agonist treatment of intact cells in culture often results in an accumulation of neutral lipids that are sometimes, visible by light microscopy, can be stained black with osmium tetroxide and red with Oil Red O (Fig. 5). This has been considered as a strong indication of PPAR-g activation (Bhandari and Schemitsch, 2002; Brodie et al., 1999; Castillo et al., 1999; Diascro et al., 1998; Gros et al., 1999; Habinowski and Witters, 2001; Harmon et al., 2002; Kim et al., 2001; Lenhard et al., 2000; Levert et al., 2002; Okazaki et al., 1999; Rivier et al., 2000; Shi et al., 2000; Starkey et al., 2003; Tang et al., 2003; Tchoukalova et al., 2000; Zehentner et al., 2000; Leesnitzer et al., 2002). Using the Red Oil O assay, a dose–response relationship for

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A 0.12 0.10 0.08 0.06 0.04 0.02 0.00 1

10 NAGly [uM]

100

B 0.19 0.17 0.15 0.13 0.11 0.09 0.07 0.05

1

10 NAGly [uM]

100

Fig. 4. Increased Oil Red assay response following treatment of RAW cells with NAGly (Burstein and Karim, unpublished data). The adipocyte differentiation assay was performed as described by Mukherjee et al. (2000). The RAW cells (American Type Culture Collection) are cultured in DMEM media supplemented with 10% calf serum. Two days after reaching confluence, cells are treated with NAGly, or vehicle (10 Al DMSO) in the presence of 10 Ag /ml insulin every other day. After 10 days of treatment with NAGly cells are fixed and stained with Oil Red O (Sigma). Vehicle treated cells were used as the baseline control.

AJA has been determined in RAW cells under the conditions described for Fig. 4 (Fig. 6; Burstein and Karim, unpublished data). These preliminary results suggest a possible biphasic relationship with AJA showing inhibition of differentiation at low concentrations and stimulation at high levels. Such biphasic effects are commonplace among cannabinoids and have also been observed with AJA. Also shown in Fig. 6 are data similarly obtained for the potent anti-

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Fig. 5. Induction of adipocyte differentiation by ajulemic acid. 3T3 L1 fibroblasts were cultured in DMEM supplemented with 10% calf serum. Two days after confluence, cells were treated with 0.1% DMSO, 20 AM AJA, or 1 AM GW347845 in the presence of 10 Ag/ml of insulin. (A) Oil Red O staining for detection of adipocyte differentiation. GW347845 is a potent PPAR g specific ligand and serves as a positive control (Cobb et al., 1998). After 7 days of treatment with GW347845 and 10 days of treatment with AJA, cells were fixed and stained with Oil Red O. Red staining indicates lipid droplets in the cytoplasm. (B) RTPCR (reverse transcriptase-polymerase chain reaction) analysis of adipocyte specific genes. PCR products were analyzed on 1% agarose gel and stained with ethidium bromide. PPAR2 and aP2 were induced significantly after treatment with 1 AM GW347845 or 20 AM AJA compared with vehicle-treated cells. The housekeeping gene GAPDH was expressed equally in all samples. (Liu et al., 2003).

inflammatory drug indomethacin. Only stimulation was observed over the limited concentration range studied.

Possible treatment of insulin resistance (Type 2 diabetes) with cannabinoids There is a growing body of evidence that the activation of PPAR-g can relieve the effects of type 2 diabetes. Drugs belonging to the category called thiazolidenediones (TZDs) are now used to treat this condition (Bell, 2003; Raji and Plutzky, 2002) and are believed to act by binding to PPAR-g (Hauner, 2002; Larsen et al., 2003; Otto et al., 2002; Van Gaal and Scheen, 2002). There are, however, limitations to their use, namely, toxic side effects that sometimes occur during chronic treatment with the TZDs (Larsen et al., 2003; Lebovitz, 2002) although this is a subject of some debate (Stumvoll, 2003). This suggests the need for novel agents namely, PPAR-g agonists that are free of toxic side effects (Gurnell et al., 2003) to address this problem.

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A

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OIL RED/DNA

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DMSO

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B 0.6 0.5 0.4 0.3 0.2 0.1 0.0 -0.1 1

10

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INDOMETHACIN [uM]

Fig. 6. Effect of concentration in the Oil Red O assay for AJA and indomethacin in RAW cells. Conditions as described in Fig. 4. (Burstein and Karim, unpublished data)

As discussed above, a recently published study (Liu et al., 2003) provides an example of a PPAR-g activator that has been shown to be well tolerated in humans (Karst et al., 2003). The compound, ajulemic acid, is a synthetic cannabinoid with analgesic and anti-inflammatory properties that is completely free of psychotropic activity. This raises the possibility that cannabinoid related molecules that activate PPAR-g may become candidates for the discovery of novel drugs to treat type 2 diabetes as well as disorders of lipid metabolism.

Acknowledgements The author is the recipient of NIH Grant DA12178.

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