Phosphodiesterase type 5 (PDE5) in the adipocyte: a novel player in fat metabolism?

Phosphodiesterase type 5 (PDE5) inthe adipocyte: a novel player in fatmetabolism?Andrea Armani1, Vincenzo Marzolla2, Giuseppe M.C. Rosano1, Andrea Fabbri3 andMassimiliano Caprio1

1 Center for Clinical and Basic Research, Scientific Institute for Research, Hospitalization and Health Care (IRCCS) San Raffaele

Pisana, Rome, Italy2 San Raffaele Sulmona, Sulmona (AQ), Italy3 Endocrinology Unit, Sant’Eugenio & CTO A. Alesini Hospital, ‘Tor Vergata’ University of Rome, Rome, Italy

Review

Phosphodiesterase type 5 (PDE5) is expressed in manytissues (e.g. heart, lung, pancreas, penis) and plays aspecific role in hydrolyzing cyclic guanosine monopho-sphate (cGMP). In adipocytes, cGMP regulates crucialfunctions by activating cGMP-dependent protein kinase(PKG). Interestingly, PDE5 was recently identified inadipose tissue, although its role remains unclear. Itsinhibition, however, was recently shown to affect adi-pose differentiation and aromatase function. This reviewsummarizes evidence supporting a role for the PDE5-regulated cGMP/PKG system in adipose tissue and itseffects on adipocyte function. A better elucidation of therole of PDE5 in the adipocyte could reveal new thera-peutic strategies for fighting obesity and metabolic syn-drome.

IntroductionAdipose tissue traditionally represents the primary site ofenergy storage. Mature adipocytes are endowed with acomplex system that is able to regulate finely the oxidationof free fatty acids and their re-esterification into triglycer-ides according to the needs of the body [1]. Over the pastseveral years the central importance of adipose tissue hasbeen increasingly recognized among researchers in thefield of metabolic disorders. Indeed, excessive expansionof adipose tissue contributes to the development of insulinresistance and diabetes, and thus to metabolic syndrome(MS) [2,3]. Adipose tissue synthesizes adipokines that,once released into the bloodstream, affect the function ofseveral tissues and organs, directly contributing to theregulation of whole-body metabolism. Therefore, the de-velopment of dysfunctional adipocytes leads to alteredfunction of downstream tissues receiving signals fromfat [1,4,5].

A large family of PDE enzymes catalyze the hydrolysisof cyclic adenosine monophosphate (cAMP) and cGMP tothe corresponding 50 nucleotide monophosphates. To date,eleven different PDEs (PDE1–11) have been characterized,and these differ in selectivity for cyclic nucleotides, sensi-tivity to inhibitors and activators, physiological roles, and

Corresponding author: Caprio, M. ([email protected]).

404 1043-2760/$ – see front matter � 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.t

tissue distribution [6]. Over the past few years severalmolecules selectively inhibiting PDE catalytic activitieshave been developed for the treatment of diverse diseases,but only PDE type 5 inhibitors (PDE5i) have reachedextensive clinical application worldwide, predominantlyfor treating erectile dysfunction and pulmonary hyperten-sion [7]. PDE5 is the principal cGMP-specific phosphodi-esterase in humans and is widely expressed in severaltissues including pancreas, skeletal muscle, smooth mus-cle, and corpus cavernosum, to name a few. In particular,its expression in lung and penile vasculature is dramati-cally more abundant than in heart [8].

Both cAMP and cGMP are involved in adipose differen-tiation and lipolysis in adipose cells [9,10]. In fact, the non-selective PDE inhibitor isobutylmethylxanthine has beenwidely used to promote adipose differentiation in vitro,confirming the importance of PDEs in controlling adipocytefunction [11]. In human adipose tissue, phosphodiesterasetype 3B (PDE3B) is known to be widely expressed [12].PDE5 has also been detected recently in human adipo-cytes, and its expression levels are modulated during adi-pose differentiation in vitro [12,13], together withphosphodiesterase type 11 (PDE11) [13], which shows highsequence homology to PDE5 [14] and acts on both cAMPand cGMP with similar Km values [6]. Interestingly, recentreports show that PDE5 inhibition with subsequent PKGactivation increases differentiation of 3T3-L1 preadipo-cytes and aromatase activity in human adipocytes[13,15]. This provides evidence for the first time thatadipocytes express functional PDE5, raising the hypothe-sis that other important aspects of fat metabolism could bemodulated by PDE5.

This review analyzes possible targets of PDE5, specifi-cally the signaling pathways affected by PKG in adipo-cytes, and explores the potential benefits of PDE5modulators in the context of adipocyte dysfunction andobesity.

Main functions of PDEs in adipocytesNumerous cell functions are regulated by the secondmessengers cAMP and cGMP. Indeed, their productionand degradation by 3’,5’-cyclic nucleotide PDEs determine

em.2011.05.004 Trends in Endocrinology and Metabolism, October 2011, Vol. 22, No. 10

Box 1. cAMP, cGMP, PKA and PKG: a complex interplay in

the adipocyte

To properly evaluate intracellular cAMP and cGMP levels it should

be considered that cyclic nucleotides regulate physiological pro-

cesses on a very short timescale (seconds to milliseconds). More-

over, both cAMP and cGMP pools are localized within discrete

subcellular compartments, and within a limited number of cells in a

tissue undergoing rapid turnover. In most tissues cAMP levels are

estimated to be in the nanomolar range, whereas GMP levels are

generally one tenth of those measured for cAMP [68]. In particular,

in primary human adipocytes cGMP is barely detectable (1.7 �0.2 pmol/100 mg lipid) whereas cAMP is more abundant (12.0 �2.0 pmol/100 mg lipid) following appropriate hormonal stimulus

[12]. Importantly, cGMP also behaves as a competitive inhibitor for

PDE3B activity in cAMP hydrolysis, suggesting reciprocal interplay

between PDE3B and PDE5 (Figure 1).

Although PKA and PKG are the principal effectors of cAMP and

cGMP, respectively, evidence for ‘cross-activation’ of PKA by cGMP,

and of PKG by cAMP, has been reported in different cell models

[69,70]. PKA and PKG show high sequence homology and their

selectivity for cAMP/cGMP might depend on only one amino acid

residue in their cyclic nucleotide binding site [71]. Therefore, high

concentrations of cGMP could lead to some degree of PKA

activation, and high levels of cAMP could increase PKG activity

[69,70]. Indeed, PKG affinity for cAMP can be increased in particular

conditions, such as autophosphorylation [72]. Few studies have

addressed the relative tissue distributions of PKG and PKA. In the

heart, PKG levels were found to be �20-fold lower than those of PKA

[8], whereas comparative quantification in adipose tissue has not

been investigated, to our knowledge.

Review Trends in Endocrinology and Metabolism October 2011, Vol. 22, No. 10

intracellular cyclic nucleotides levels. Distinct PDEs mod-ulate specific signal transduction pathways, playing im-portant roles in several cell functions [6]. Furthermore,cAMP-dependent protein kinase (PKA) and PKG representadditional downstream effectors of cAMP and cGMP, re-spectively (Box 1). PDE3B is the most abundant PDE inhuman adipocytes [12] and shows affinity for both cyclicnucleotides, although with a different Km from PDE5 [6].PDE3B primarily controls intracellular cAMP levels inadipocytes (Figure 1), where its activity is specificallycontrolled by insulin, through AKT phosphorylation.PDE3B also inhibits lipolysis in differentiated fat cells[10,16]. Intracellular cAMP is widely known as a powerfulinducer of adipogenesis through specific PKA activationand subsequent phosphorylation of cAMP-responsive ele-ment binding protein (CREB), which induces transcriptionof adipogenic genes [17,18]. A brilliant report by Reuschet al. showed that CREB transactivation by an adipogeniccocktail was both necessary and sufficient to induce adi-pogenesis in 3T3-L1 cells [18]. However, in vitro studies inbaby hamster kidney (BHK) cells [19] and in vascularsmooth muscle and neuronal cells [20] demonstrated thatCREB can also be phosphorylated by cGMP-dependentmechanisms. Accordingly, lack of PKG in brown adipocytesresults in impaired CREB phosphorylation, together withsevere disruption of differentiation and mitochondrial bio-genesis [21].

Taken together, these data underscore the significanceof intracellular cGMP in regulating many functions of theadipocyte.

Given the high binding capacity and specificity of PDE5for cGMP, interest in its potential roles in adipocyte phys-iology has grown in recent years. PDE5 mRNA expression

was first detected in human subcutaneous adipose cells byMoro et al., where its transcript levels are less abundantthan those of PDE3B [12]. Interestingly, the highest levelsof PDE5 protein were detected in preadipocytes and theselevels decreased during adipose maturation [12]. We re-cently confirmed a similar pattern of expression of PDE5transcripts during adipose differentiation of human viscer-al preadipocytes [13]. We also characterized the expressionpattern of PDE11, and observed an opposite expressionprofile during adipocyte maturation where the highesttranscript levels were found in terminally differentiatedadipocytes [13]. Because PDE11 transcript levels in ma-ture adipocytes are higher than those of PDE5, we cannotexclude a preponderant physiological role of PDE11 indifferentiated fat cells. In particular, given its ability tohydrolyze both cyclic nucleotides, PDE11 might regulateboth cAMP- and cGMP-mediated lipolysis. Although thephenotype of PDE11-deficient mice suggests an involve-ment in spermatogenesis [6], adipocyte function in thismodel has not been yet examined. We cannot exclude thatlack of PDE11 in adipocytes might reveal its potentialphysiological relevance in modulating adipocyte function,especially in processes known to be regulated by both cyclicnucleotides, such as lipolysis.

Potential role for PDE5 in adipocyte differentiation andmitochondrial biogenesisA role for PDE5 in adipogenesis was recently shown by theobservation that PDE5 blockade during 3T3-L1 preadipo-cyte differentiation increased intracellular lipid dropletsas well as expression of adipocyte-specific genes peroxi-some proliferator activated receptor-g (PPARg), fatty acidsynthases (FAS), and adiponectin. Unfortunately, PDE5mRNA or protein expression in this cell model was notcharacterized [15], raising the question of the specificity ofthe effects of sildenafil in this cellular model. Similarresults were obtained with the cGMP analog 8-pCPT-cGMP [8-(4-chlorophenylthio)guanosine-3’,5’-cyclic mono-phosphate]. Interestingly, co-treatment with the PKG in-hibitor Rp-8-pCPT-cGMP blocked the effects of both PDE5inhibition and the cGMP analog on triglyceride accumula-tion, suggesting that PKG activation is necessary to medi-ate the effects of increased cGMP levels in promoting theadipogenic process [15]. In accordance with these data,atrial natriuretic peptide (ANP) positively regulates thedifferentiation of rat preadipocytes through an increase incGMP and subsequent PKG activation [22]. Brown andwhite adipocytes represent two different cell phenotypesinterspersed in the context of adipose tissue [23]. Consid-ering that these two distinct cell types share commondifferentiation pathways, the brown adipocyte representsa valuable tool for studying the role of PKG in adiposetissue. Indeed, convincing evidence supporting the role ofcGMP and PKG in adipogenesis has been shown in brownadipocytes: immortalized primary brown pre-adipocytesisolated from PKG knockout (KO) mice displayed a reducedcontent of lipid droplets, decreased expression of PPARg,CCAAT/enhancer binding protein-a (C/EBPa) and adipo-cyte fatty acid-binding protein (aP2), together with in-creased activity of RhoA [21], a small monomeric G-protein involved in the regulation of several cell processes

405

LIPID DROPLET

NU

CLE

US

PKGPDE3B

PDE5

PKA

NPR-A

PDE5i

CATECHOLAMINES INSULIN NATRIURETIC FACTORS(ANP, BNP)

ADIPOSE CELL

β- adrenergic

receptor

(a)

(b)

(c)

(d)

(e)

(f)

(g)

5’AMP

cAMP

cGMP

5’GMP

P

NO

NPGC

IR

AC

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sGC

Figure 1. Crosstalk between cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) in the adipocyte. Catecholamines determine an

increase in intracellular cAMP, a well-known activator of cAMP-dependent protein kinase (PKA) (a). Intracellular levels of cAMP are primarily modulated by

phosphodiesterase type 3B (PDE3B), which is highly expressed in the adipocyte and hydrolyzes cAMP to 5’-AMP (b). Activity of PDE3B is increased by the anti-lipolytic

hormone insulin. Phosphodiesterase type 5 (PDE5) specifically hydrolyzes cGMP to 5’-GMP (c). cGMP levels influence PKG activity (d) and its downstream signaling

pathways. Moreover, PDE5 phosphorylation by PKG (e) increases PDE5 activity and represents a negative feedback mechanism that lowers PKG activity through reduction

of cGMP. Atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP), through interaction with natriuretic peptide guanylate cyclase receptor of the A subtype (NPR-

A), stimulate cGMP formation (f). Activation of soluble guanylate cyclase (sGC) by NO stimulates cGMP formation (f), which in turn inhibits PDE3B (g), finally increasing

cAMP levels. Therefore PDE5 inhibition can lead both to cGMP and cAMP increase.

Review Trends in Endocrinology and Metabolism October 2011, Vol. 22, No. 10

[24]. Interestingly, expression of a dominant negativeRhoA in PKG–/– brown adipocytes restored the ability ofthe adipocyte to accumulate triglycerides [21]. Such evi-dence confirms a central role for PKG in controlling adiposedifferentiation, also in the context of brown fat, suggestinga role for PDE5 also in controlling brown adipocyte matu-ration.

Rho-associated kinases (ROCKs) mediate several down-stream effects of RhoA, and are known to regulate a widerange of cell functions (proliferation, apoptosis, contrac-tion). Alterations of RhoA/ROCK have been observed incardiovascular diseases [25]. A role for ROCK has beendemonstrated in primary brown pre-adipocytes isolatedfrom PKG KO mice because its pharmacological inhibitionand gene silencing restored lipid accumulation and expres-sion of adipose markers. This suggests that RhoA/ROCKrepresents the final downstream effector of PKG activity inthe control of adipogenesis [21]. Mitochondrial content andmarkers of mitochondriogenesis, such as uncoupling pro-tein 1 (UCP-1) and PPARg coactivator-1a (PGC-1a), werealso reduced in PKG–/– brown adipocytes, and were re-stored by ROCK blockade [21]. All these data stronglyindicate the importance of the cGMP/PKG system in adi-pogenesis and mitochondrial biogenesis, two major aspectsof brown adipocyte physiology.

Nitric oxide (NO) is a well-known inducer of adipogen-esis in brown and white adipocytes, and NO donatingagents are known to stimulate mitochondriogenesisin differentiating brown preadipocytes [26,27]. NO also

406

activates soluble guanylate cyclase (sGC), which in turnstimulates PKG activity through increased levels of cGMP[28]. Given the importance of PDE5 in regulating theoverall abundance of cGMP and the central role of cGMPin controlling adipogenesis, it seems likely then that PDE5regulates adipogenesis.

Several members of the Wnt (wingless-type MMTVintegration site) family have been shown to inhibit adipo-genesis [29]. For example, Wnt10b increases the stabilityof b-catenin in the cytoplasm, preventing it from translo-cating to the nucleus and activating T cell factor/lympho-cyte enhancer factor (TCF/LCF) proteins; therefore,Wnt10b ultimately represses adipogenic genes [29]. Im-portantly, PKG activity reduces b-catenin expression andits interaction with TCF/LCF family of transcription fac-tors in colon cancer cells, with consequent inhibition ofgene target expression [30]. Moreover, in the same model,PKG promotes nuclear translocation of forkhead box O4(FOXO4) which binds to b-catenin, similarly inhibiting itsinteraction with TCF. If this phenomenon occurs also inpreadipocytes, we speculate that PKG could attenuateWnt/b-catenin pathway, together with downstream regu-lated genes, to favor adipogenesis (Figure 2). In support ofthis hypothesis, translocation of FOXO4 has been found topromote lipid droplet accumulation in 3T3-L1 cells, fattyacid biosynthesis, and glucose uptake [31]. Therefore, Wntsignaling repression represents another potential target ofthe PDE5�cGMP�PKG system in controlling adipose dif-ferentiation.

LIPID DROPLET

HSL

sGCGC

ARO

ANPBNP

NPR-A

RhoA

ROCKAKT

IR

p38

CREB

PDE5

PKG

FFA

FOXO4

NUCLEUS

TCF

aromatase

adiponectin

adipogenic genes

β-CATENIN

Testosterone

Estradiol

INSULIN

ADIPOSE CELL

(a)(b)

(c)

(g)

(e)

(f)

(d)

(h)

?

?

?cGMP

5’-GMP

NOADIPONECTIN

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Figure 2. cGMP-dependent protein kinase (PKG) regulates important metabolic processes in the adipocyte. PKG activity regulates adipocyte lipolysis, differentiation, and

adipokine secretion. Activation of either soluble guanylate cyclase (sGC) or natriuretic peptide guanylate cyclase receptor of the A subtype (NPR-A) increases cyclic guanosine

monophosphate (cGMP), which stimulates PKG (a). PKG phosphorylates and activates hormone sensitive-lipase (HSL) (b), which hydrolyzes triglycerides into non-esterified

fatty acids (FFA) and glycerol. PKG plays an important role in stimulating adipogenesis, through activation of cAMP-responsive element binding protein (CREB) and p38 MAP

kinase (c), leading to induction of adipogenic genes. PKG potentiates insulin signaling by reducing inhibitory phosphorylation of insulin receptor substrate 1 (IRS-1) by Rho-

associated kinase (ROCK) (d). PKG inhibits ROCK activity, thereby favoring the association of phosphoinositide 3-kinase (PI3K) with IRS-1, finally increasing Akt activation (e) and

adipogenesis. PKG might affect b-catenin-mediated gene expression in adipocyte via its influence on FOXO4 (f). PKG upregulates adiponectin expression (g), with potential

effects on insulin sensitivity. PDE5 inhibition upregulates aromatase (ARO) in human adipose tissue (h), with increased estrogen release.

Review Trends in Endocrinology and Metabolism October 2011, Vol. 22, No. 10

Activation of p38 MAP kinase promotes human adipo-genesis through phosphorylation of the early adipogenictranscription factor CCAAT/enhancer binding protein-b(C/EBPb), whereas its pharmacological blockade inhibitsadipocyte differentiation [32]. Differently, blockade of ei-ther extracellular signal-regulated kinases (ERKs) or c-Jun amino-terminal kinases (JNKs) has no effect on adi-pogenesis [32]. In brown murine adipocytes, Hass et al.showed that p38 activation requires PKG activity [21].Therefore, PDE5 inhibition might stimulate C/EBPb

transactivation and adipogenesis through an increase inPKG and p38 activity (Figure 2). In brown adipocyteslacking PKG, CREB activation is reduced, suggesting thatPKG is important for CREB activation [21]. As alreadydiscussed in the introduction, CREB favors adipogenesisdue to its ability to bind cAMP-responsive sequence ele-ments (CREs) within the promoters of several adipocyte-specific genes [18]. Increased intracellular cGMP stimu-lates CREB phosphorylation in several types of cells[19,33–35], and it therefore seems likely that PKG canalso directly activate CREB in adipose tissue (Figure 2).Finally, it is possible that the downregulation of PDE5expression observed during the course of adipose conver-sion [12,13] contributes to maintaining higher levels ofcGMP, with a concomitant increase in PKG activity, finallyfavoring the differentiation process (Figure 2).

PDE5 involvement in insulin signalingPKG influences insulin signaling in brown adipocytesthrough ROCK involvement, as discussed. In brown pre-

adipocytes lacking PKG, increased ROCK activity leads toelevated insulin receptor substrate 1 (IRS-1) Ser 636/639phosphorylation resulting in decreased association of phos-phoinositide 3-kinases (PI3K) with IRS-1 and reduced Aktactivation [21] (Figure 2). Accordingly, expression of aconstitutively active form of Akt in PKG–/– brown preadi-pocytes recovered defective mitochondrial biogenesis andadipogenesis [21]. Therefore, the attenuation of insulinsignaling appears to contribute to defective adipogenesisand mitochondrial biogenesis, both of which are promotedby insulin [36,37] in PKG–/– brown adipocytes.

Glucose transporter type 4 (Glut4) is a protein respon-sible for insulin-regulated glucose transport and translo-cation into the cell, primarily in fat and muscle [38]. Insulininduces Glut4 translocation to the plasma membrane andsubsequent glucose uptake predominantly in heart, skele-tal, vascular smooth muscle cells (VSMCs) and adiposecells [38–40]. NO increases sGC activity and in turn ele-vates cGMP levels [41]. Importantly, insulin stimulatesGlut4 translocation and glucose uptake in VSMCs, throughNO production and sGC activation, with subsequentincreases of cGMP levels and PKG activity [42], suggestinga direct link between glucose uptake and PKG activation.In human umbilical vein endothelial cells (HUVECs) werecently showed that PDE5 inhibition increases eNOSactivity and NO production [43]. Interestingly, PDE5blockade stimulates insulin-mediated glucose uptake in3T3-L1 preadipocytes [15]. Such effects might be due to theincrease in NO production, with subsequent higher levelsof cGMP and increased PKG activity, and could occur in fat

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tissue, given that the entire enzymatic machinery of theNO system, including sGC, is expressed and functional inthe adipocyte [44]. Evidence in vivo derived from miceoverexpressing PKG in adipose tissue confirm the rele-vance of PKG in fat metabolism; in fact, PKG transgenicmice fed a high-fat (HF) diet for 16 weeks displayeddecreased body weight gain, lower body-fat mass, andimproved glucose tolerance compared to HF wild-typecontrols as a result of increased lipolysis and thermogene-sis [45]. Surprisingly, such effects were present only infemale transgenic mice, and not in males, probably due todifferent levels of circulating estrogens that are known toenhance cGMP/PKG signaling [46]. Importantly, involve-ment of PDE5 in insulin signaling has been confirmed invivo because chronic treatment with the PDE5 inhibitorsildenafil in a mouse model of diet-induced insulin resis-tance improved insulin sensitivity, shown as a reduction infasting insulin and glucose levels [47], concomitant with anincrease in arterial cGMP levels.

Potential role for PDE5 in lipolysisLipolysis leads to the release of fatty acids into the blood-stream where they provide fuel for tissues requiring energy[48]. Regulation of lipolysis is a crucial function in adipo-cyte metabolism, and altered lipolysis can contribute toobesity [49]. In primates, lipolysis is stimulated by cate-cholamines as well as by natriuretic peptides (NPs) [10]. Inparticular, ANP and brain natriuretic peptide (BNP) acti-vate the NP guanylate cyclase receptor of the A subtype(NPR-A) localized to the surface of adipose cells (Figure 2).Activated NPR-A increases intracellular levels of cGMPthat then activate PKG, which in turn phosphorylateshormone sensitive-lipase (HSL). Once activated, HSLhydrolyzes the triglycerides in non-esterified fatty acidsand glycerol. In general, ANP is not an efficient inducer oflipolysis in non-primate adipocytes because of scarce ex-pression of NPR-A [50], although NPR-A is expressed incultured rat adipocytes, suggesting potential involvementin lipolysis induction [22]. ANP induces lipolysis through acGMP-specific pathway distinct from that activated bycatecholamines, which increase cAMP levels and PKAactivity [10]. However, both pathways converge on activa-tion of HSL and lipolysis induction. As discussed previous-ly, adipocyte cAMP degradation is principally regulated byPDE3B whose activity is increased by the anti-lipolytichormone insulin [10,16,49]. PDE3B hydrolyzes cAMP, andnegatively modulates catecholamine-induced lipolysis, butdoes not significantly influence ANP-mediated lipolysisthat relies on cGMP generation [10]. In consideration ofthis complex body of evidence, intracellular levels of bothcyclic nucleotides are crucial to the regulation of the rate oflipolysis.

Important elements of PDE5 physiology have beenclarified by pharmacological studies with specific inhibi-tors. Surprisingly, PDE5 inhibition in human subcutane-ous adipocytes resulted in a small but significant increasein intracellular cGMP levels, but without any net effect onlipolysis rate [12], as one could have predicted. We con-firmed these results in visceral adipocytes [51]. However,no molecular mechanism was proposed to explain the lackof an effect of cGMP increase on lipolysis [12]. It might be

408

speculated that the magnitude of the increase in cGMPlevels due to PDE5 inhibition is too small to activate PKGand promote lipolysis. Owing to the lack of animal modelslacking or overexpressing PDE5 in fat tissue, the relevanceof PDE5 function in lipolysis is currently unknown in vivo.Further analyses are required to define better this impor-tant issue in the context of adipocyte metabolism.

Potential PDE5 involvement in adiponectin productionAdipokine and cytokine secretion represents an importantfunction of fat, and this is crucial for the integration ofadipose tissue in whole-body homeostasis [2]. Obesity anddysregulation of adipokine secretion have been implicatedin the development of type 2 diabetes, hypertension andcardiovascular disease [4]. Compared to lean subjects,obese individuals have lower circulating levels of natri-uretic factors (NPs) [52]. Reduced levels of NPs, that areimportant modulators of lipolysis as discussed, could pro-mote lipid accumulation in adipose tissue, thereby favoringthe development of visceral adiposity that is in turn asso-ciated with dyslipidemia, insulin resistance, and increasedcardiovascular risk [53]. This observation suggests bidirec-tional crosstalk between the cardiovascular system and fattissue, mediated by cardiomyocyte-produced NPs that af-fect adipocyte metabolism. Indeed, adiponectin secretion isin part controlled by cardiac NPs [54]. Treatment of humanprimary subcutaneous adipocytes with ANP and BNPincreased expression and secretion of adiponectin, awell-known insulin-sensitizing and anti-inflammatory hor-mone secreted by adipocytes, and whose synthesis is re-duced in obesity, insulin resistance and MS [4,54,55].Accordingly, the reduced levels of circulating NPs in obeseindividuals might explain, at least in part, the observedlow levels of circulating adiponectin in obese subjects.Importantly, co-treatment of human adipocytes with aPKG inhibitor suppresses adiponectin secretion [54], sug-gesting that expression of adiponectin takes place viaactivation of cGMP/PKG system, and that it could poten-tially be regulated by PDE5 activity (Figure 2). Futurestudies focused on the long-term effects of PDEi on circu-lating levels of adiponectin and glucose metabolism couldprovide further insights on the functional link betweenPDE5 and adiponectin in vivo.

PDE5 involvement in aromatase expressionCytochrome P450 CYP19, also known as aromatase (ARO),is principally expressed in ovary and adipose tissue andconverts androgens to estrogens [56]. The role of adiposetissue as a valuable source of 17b-estradiol (E2) derivingfrom aromatization of androgens, is of central importancein men and postmenopausal women [57]. In adipose cells,ARO expression is induced by prostaglandin E2 (PGE2)through the activation of both protein kinase C (PKC) andPKA [58]. We recently showed that ARO expression andactivity are upregulated by pharmacological PDE5 inhibi-tion in primary human visceral adipocytes, with a conse-quent increase in estrogen production [13]. The signalingpathway responsible for the increase in CYP19 transcrip-tion remains controversial, although a potential regulatorymechanism linking PDE5 inhibition with increased CYP19transcriptional activity is suggested by the identification of

Review Trends in Endocrinology and Metabolism October 2011, Vol. 22, No. 10

two distinct CRE-like sequences in the promoter of theCYP19 gene that can be bound by CREB [59]. Althoughthese data describe a prevalent role of cAMP in AROregulation, we cannot exclude the possibility that CREB-driven CYP19 transcription could be activated indirectlyby PKG/cGMP subsequent to PDE5 inhibition (Figure 2).In our recent work we used tadalafil (that blocks bothPDE5 and PDE11) and sildenafil (PDE5-specific) to blockPDE5 [13]. PDE11 is abundantly expressed in matureadipocytes and could represent an additional target oftadalafil. Importantly, we observed similar effects onARO expression using either pharmacological compound,confirming the specific involvement of PDE5. Moreover,treatment with milrinone, a specific inhibitor of PDE3B,did not affect ARO expression, thereby excluding a role ofPDE3B in ARO regulation [13]. Of course, the physiologicalrelevance of the effects described in human visceral adi-pocytes requires further in vivo studies.

Under physiological conditions, estrogen affects multi-ple body tissues [60]. Estrogen receptors (ERa and ERb)are expressed in vascular smooth muscle and endothelialcells (ECs) where they mediate both the rapid non-genomicand genomic cardiovascular effects of estrogens, mainlyimproving vasodilation [13,61]. Estrogens display vasopro-tective and anti-inflammatory effects and have been shownto exert beneficial effects on the cardiovascular systemunder some conditions [56,61,62]. The effects of PDE5

Corpus Cavernosum

Vascular relaxation and erection

PDE5

Pulmonary vascu lar system

Vascular relaxation,improvement of pulmonary hypertension

Improvement of

Heart

cardiomyoc yte stiffness and contractility

PDE

Figure 3. Tissue targets and major clinical effects of PDE5 inhibitors (PDE5i). PDE5i incre

relaxation. PDE5i were originally developed to treat angina pectoris, and their use is cur

failure, cardiac hypertrophy, where they improve myocardial stiffness and contractility. I

(eNOS) activity and NO production, which stimulates Glut4 translocation and glucose u

after treatment with PDE5i. Finally, given that PDE5 is expressed in human adipose tissu

potential.

inhibition on adipocyte ARO activity, by means of a tran-sient increase in E2 concentrations in perivascular fat,might improve endothelial function, representing a sup-plemental mechanism responsible for the rapid vasodilatorresponses of the penile and systemic arteries in response toPDE5 inhibitors. ARO-deficient male mice develop glucoseintolerance and insulin resistance [60] and several aspectsof the loss of ARO recapitulate alterations occurring inpatients with MS [56]. Adipose tissue express both ERs[63] and in vivo studies show that lack of ERa produces amarked increase in white adipose tissue and insulin resis-tance in mice of both sexes [64]. All this evidence highlightsthe importance of ER signaling in different tissues, includ-ing fat, to regulate cardiovascular function and energyhomeostasis, and suggests new and unexpected therapeu-tic applications of PDE5 inhibitors in improving endothe-lial and adipocyte function.

Concluding remarksPDE5 represents a physiological counter-regulatory mech-anism to modulate the amplitude and the duration ofcGMP signaling; hence its inhibition has become a valuablepharmacological tool in several systems. PDE5 inhibitorsare widely used in erectile dysfunction, where their intro-duction revolutionized the field of sexual medicine, andmore recently in pulmonary arterial hypertension [65]. Theuse of these molecules is currently under investigation for

Adipose tissue

Increased aromatase activity, adipogenesis,and insulin sensitivity

i

MuscleImprovement of insuli n sensitivity

Increased eNOS activity, improved insulin

PI3KAKT

Endothelial cell

signalinginsulin

eNOS NO

5

TRENDS in Endocrinology & Metabolism

ase cGMP in vascular smooth muscle cells (VSMCs) to influence systemic vascular

rently studied for many aspects of cardiovascular disease, including diastolic heart

n endothelial cells, PDEi enhance insulin signaling, endothelial nitric oxide sinthase

ptake. Such effects might explain the improvement in insulin sensitivity observed

e, application of PDEi in the treatment of obesity and related metabolic disease has

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Box 2. Outstanding questions in the field

Data confirming a role for PDE5 in adipocyte biology in vitro have

been recently reported [13,15,21]. However, a better understanding

of the complex role of PDE5 in fat metabolism and whole body

homeostasis requires the use of transgenic animal models either

lacking or overexpressing PDE5 in adipose tissue. This will clarify

the role of PDE5 in adipose expansion and metabolism, and also in

glucose homeostasis and vascular function in vivo.

Analysis of expression and activity of PDE5 in different sites of

adipose tissue (i.e. visceral vs subcutaneous), and also in diverse

metabolic conditions (i.e. high-fat diet vs low calorie intake) could

reveal if PDE5 can be considered to be a reliable ‘marker’ of

metabolic dysfunction of the adipocyte. Importantly, chronic treat-

ment with the PDE5 inhibitor sildenafil in a mouse model of diet-

induced insulin resistance caused a significant improvement in

insulin sensitivity [47]. However, the efficacy of long-term treatment

with PDE5i awaits demonstration in human metabolic diseases such

as obesity and insulin resistance. Indeed, if these studies confirm a

role for PDE5 in fat physiology and glucose metabolism, clinical use

of selective PDE5 modulators could become a promising pharma-

cological tool to fight MS.

Review Trends in Endocrinology and Metabolism October 2011, Vol. 22, No. 10

many aspects of cardiovascular medicine, including treat-ment of diastolic heart failure [66] and cardiac hypertro-phy, where PDE inhibitors have been shown to improvemyocardial stiffness and contractility [67] (Figure 3). In allthese cases, PDE5 inhibitor effects are accomplished byenhancing the downstream effects of NO in VSMCsthrough reduced Ca2+ concentration and subsequent relax-ation and vasodilation. Given the wide expression of PDE5in several tissues outside of the cardiovascular system,interest in different potential applications of PDE5i isincreasing.

PDE5, via its effects on cGMP concentrations, is animportant modulator of PKG activity in the adipocyteand controls crucial functions of the adipocyte via PKG,such as differentiation, mitochondrial biogenesis and hor-mone release. As discussed, PDE5 blockade in adiposetissue also increases local estrogen conversion, resultingin potential protective effects on vasculature and endothe-lial function (Figure 3). This could also represent an auto-crine mechanism capable of controlling the expansion of fattissue, thereby preventing its metabolic dysfunction.

Given the importance of adipose tissue as a strategicorgan in the context of energy homeostasis, a better eluci-dation of the role of PDE5 and its pharmacological inhibi-tion could help to clarify the pathophysiology of obesity andMS. Indeed, PDE5 emerges as a novel molecular effectorthat is able to influence several functions of adipocytes and,as such, could become a promising target in the design ofnew therapies to fight MS (Box 2).

AcknowledgmentsThe authors wish to thank Richard Karas and Caterina Mammi forcritical reading of the manuscript and invaluable suggestions. This workwas supported by institutional funding from IRCCS San Raffaele Pisanaand the University Tor Vergata (Progetti Ricerca Interesse NazionaleMinistero dell’Universita e della Ricerca, 2007) to A.F.

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