Metabolic Regulation in Adipocytes by Prostanoid Receptors

Abstract

Prostanoids are a group of typical lipid mediators that are biosynthesized from arachidonic acid by the actions of cyclooxygenases and their subsequent terminal synthases. Prostanoids exert a wide variety of actions through their specific membrane receptors on target cells. In addition to their classical actions, including fever, pain, and inflammation, prostanoids have been shown to play pivotal roles in various biological processes, such as female reproduction and the maintenance of vascular and gut homeostasis. Moreover, recent research using mice deficient in each of the prostanoid receptors, or using agonists/antagonists specific for each receptor clarified novel actions of prostanoids that had long been unknown, and the mechanisms therein. In this review, we introduce recent advances in the fields of metabolic control by prostanoid receptors such as in adipocyte differentiation, lipolysis, and adipocyte browning in adipose tissues, and discuss the potential of prostanoid receptors as a treatment target for metabolic disorders.

1. INTRODUCTION

Prostanoids, comprising of four types of prostaglandins (PGs; PGD₂, PGE₂, PGF_2α, and PGI₂) and thromboxane (TX) A₂, are a group of lipid mediators synthesized from arachidonic acid (AA) by the action of cyclooxygenases (COXs) as the rate-limiting enzyme.^1,2) Non-steroidal anti-inflammatory drugs, such as aspirin and indomethacin, exert antipyretic, analgesic, and anti-inflammatory effects by suppressing the biosynthesis of these lipid mediators. Prostanoids exert a wide variety of actions by acting on membrane-bound G protein-coupled receptors (GPCRs) that are specific for each prostanoid, and have been conventionally considered to work as inflammatory mediators.^3,4) Recent research on prostanoid-synthesizing enzymes and prostanoid receptors using pharmacological and genetic approaches demonstrated that prostanoids elicit both pro-inflammatory and anti-inflammatory actions in a context-dependent manner, by playing crucial roles not only in the inflammatory process, but also in various physiological processes, such as female reproduction, and the maintenance of vascular and gut homeostasis.^5–9) In this review, focusing on the biological actions of prostanoids in adipocytes, which play a central role in glycolipid metabolism, we summarize recent advances in the physiological regulation of white and brown adipocytes by prostanoid receptors. We also discuss the possibility of prostanoid receptors as therapeutic targets of metabolic disorders, including obesity, type II diabetes, and fatty liver.

2. PROSTANOID SYNTHESIS AND PROSTANOID RECEPTORS

Prostanoids are synthesized by three sequential enzymatic reactions. The first reaction is AA release from membrane phospholipids by the action of phospholipase A₂ (PLA₂). PLA₂ molecules such as cytosolic PLA₂α (cPLA₂α) have long been considered to be solely responsible for the supply of AA to COX enzymes.¹⁰⁾ Recently, it was shown that PLA₂-independent AA-producing pathways also contribute to prostanoid synthesis. In the brain, monoacylglycerol lipase (MAGL), which releases AA from 2-arachidonoylglycerol (2-AG), participates in fever generation and the development of neurodegenerative diseases.^11,12) Alternatively, adipocyte triglyceride lipase (ATGL) supplies AA for prostanoid synthesis by cleaving triglycerides (TGs); this type of AA supply was reported in several types of immune cells that penetrate into peripheral tissues, and contain lipid droplets as they cannot obtain energy from the circulation.^13,14) The second step is the conversion of AA into PGH₂ by two types of COX isozymes, COX-1 and COX-2. COX-1 is constitutively expressed in most cell types, whereas COX-2 expression is induced by various stimuli. The third reaction is the rapid conversion of unstable PGH₂ into biologically active prostanoids by each prostanoid-specific terminal synthase (e.g., microsomal prostaglandin E synthase-1 (mPGES1) for PGE₂ synthesis) (Fig. 1).

Fig. 1. Prostanoid Synthesis Pathways and Prostanoid Receptors

2-AG, 2-arachidonoylglycerol; TG, triglyceride; MAGL, monoacylglycerol lipase; ATGL, adipocyte triglyceride lipase.

Prostanoids elicit their versatile actions by binding to specific GPCRs expressed in neighboring cells. PGF_2α, PGI₂, and TXA₂ each act on a single receptor, namely, the FP receptor, IP receptor, and TP receptor, respectively. PGE₂ exerts multiple biological actions via four subtypes of PGE receptors, namely, EP1, EP2, EP3, and EP4. The DP1 receptor has long been regarded as the only receptor for PGD₂. However, in 2001, Hirai et al. reported the presence of a second receptor, DP2, which was originally named chemoattractant receptor-homologous molecule on Th2 cells (CRTH2), and belongs to an evolutionally different receptor family.¹⁵⁾ Each of the prostanoid receptor types and subtypes is coupled to a distinct intracellular signaling pathway; i.e., DP1, EP2, EP4, and IP receptors stimulate adenylyl cyclase via the Gs protein, and in contrast, EP3 and DP2 receptors are coupled to the inhibition of adenylyl cyclase via Gi protein. EP1, FP, and TP receptors elicit intracellular Ca²⁺ mobilization via Gq protein. Moreover, EP2 and EP4 receptors have been shown to induce the activation of phosphoinositide-3 kinase via the β-arrestin pathway.^16,17)

3. CLASSIFICATION OF ADIPOSE TISSUES AND THEIR FUNCTIONS

Adipose tissues are classified into white adipose tissue (WAT) and brown adipose tissue (BAT), and their component adipocytes are characteristically quite different.¹⁸⁾ White adipocytes store excess energy as TGs in a single large lipid droplet, and supply stored energy to other tissues by hydrolyzing TGs in accordance with energy needs. In addition to its role in energy storage, WAT also acts as an endocrine organ, and contributes substantially to systemic metabolic regulation by secreting a variety of intercellular signaling molecules, called adipokines, such as adiponectin, leptin, and tumor necrosis factor-α (TNF-α).¹⁹⁾ Severe obesity by excessive caloric intake induces chronic inflammation in WAT, which is often accompanied by the infiltration of macrophages and immune cells into WAT. Inflammatory cytokines and free fatty acids released from WAT are crucial risk factors for the development of insulin resistance and obesity-associated metabolic disorders, including diabetes, fatty liver, and arteriosclerosis.²⁰⁾

Brown adipocytes contain small multilocular lipid droplets and a high density of mitochondria, which contribute to their brown color.²¹⁾ The primary function of these cells is a heat production. The cells highly express uncoupling protein 1 (UCP1), which converts energy to heat without generating ATP in mitochondria. BAT is abundant in small rodents and newborns, and helps them to maintain their body temperature by nonshivering thermogenesis. Whereas small mammals, such as mice retain their BAT throughout their lifetime, BAT has been considered to disappear in adult humans. However, in 2009, several research groups reported the existence of UCP1-positive functional brown-like adipocytes in adult human WAT.^22–24) These cells are recruited to WAT by particular stimuli, including cold stress, and they are called beige or brite (brown in white) adipocytes.²⁵⁾ Many researchers considered that inducing the recruitment of these adipocytes, namely adipose tissue browning, promotes systemic energy expenditure and improves a wide range of metabolic disease symptoms.^26,27) Such background information indicates that understanding the molecular mechanism underlying the regulation of WAT function and browning is very important for the development of effective strategies for the prevention or treatment of metabolic disorders.

4. REGULATION OF ADIPOGENESIS AND ADIPOCYTE FUNCTION BY PROSTANOID RECEPTORS IN WAT

Adipocytes originate from mesenchymal stem cells (MSCs), and numerous studies on the regulation of adipogenesis have been performed using an in vitro differentiation system comprising isolated MSCs or preadipocyte cell lines, such as 3T3-L1 cells. Prostanoids were originally identified as a potential regulator of adipogenesis. The PGI₂-IP pathway was reported to exert proadipogenic actions. Activation of the IP receptor by agonists was suggested to enhance fat storage during the maturation phase of adipocyte cultures of ob1771 or 3T3-L1 cells.^28,29) Moreover, diet-induced obesity (DIO) was suppressed in mice deficient in PGI synthase or the IP receptor.^30,31) On the other hand, the PGF_2α-FP pathway was reported to attenuate adipocyte differentiation in 3T3-L1 cells and MSCs.^32,33) Indeed, mice deficient in the PGF synthase gene, Akr1B7, showed decreased PGF_2α levels in WAT in association with more severe DIO and more serious insulin resistance.³⁴⁾ Interestingly, PGF_2α analogs, such as bimatoprost and latanoprost, which are clinically used for glaucoma, have been reported to induce the atrophy of orbital fat.³⁵⁾ Alternatively, Tsuboi and Inazumi et al. found that PGE₂ also inhibits the early phase of adipocyte differentiation via the EP4 receptor in 3T3-L1 and mouse embryonic fibroblasts (MEFs).^36,37) Moreover, it was recently reported that the EP3 receptor also has anti-adipogenic potential.³⁸⁾ However, there are no reports to date directly showing irregular adipocyte numbers in WAT from these receptor-deficient mice, and therefore, it is unknown whether prostanoid receptors physiologically contribute to the regulation of adipocyte differentiation.

White adipocytes control their size and whole-body energy balance by lipogenesis and lipolysis depending on the energy situation. Lipolysis is induced in situations of energy shortage, such as in fasting or cold conditions, and epinephrine and glucagon have been identified as key hormones that promote lipolysis in these conditions. PGE₂ has also been suggested to participate in the regulation of lipolysis. PGE₂ has long been thought to work as an antilipolytic substance, presumably via the EP3 receptor, from pharmacological experiments.³⁹⁾ Jaworski et al. demonstrated that the lack of the adipocyte-specific PLA₂ AdPLA reverses DIO by increasing lipolysis.⁴⁰⁾ However, EP3-deficient mice did not show reduced DIO as observed in AdPLA-deficient mice^41,42) Further studies using adipocyte-specific EP3-knockout mice are hence required to clarify the physiological contribution of this receptor to the regulation of lipolysis. Inazumi et al. recently found that the PGE₂-EP4 pathway promotes basal lipolysis, utilizing global and adipocyte-specific EP4-deficient mice.⁴³⁾ In this study, the authors found that PGE₂ synthesis in WAT is up-regulated after food intake in an ATGL/COX-1/mPGES1-dependent manner, and that the PGE₂-EP4 pathway sustains basal lipolysis and controls fat distribution by increasing the expression or activity of lipolytic rate-limiting enzymes, such as ATGL and hormone sensitive lipase (HSL), as a negative feedback of insulin signaling (Fig. 2). EP4 deficiency results in increased adiposity by maintaining high insulin sensitivity and low hepatic fat deposition; EP4-deficient mice demonstrate a ‘metabolically healthy obesity’-like phenotype. Furthermore, the authors identified a human single nucleotide polymorphism associated with the lower expression of EP4, and demonstrated that lower EP4 expression is associated with lower prevalence of non-alcoholic fatty liver disease (NAFLD) in humans. In accordance with this finding, Shen et al. reported that habitual users of aspirin show lower NAFLD prevalence.⁴⁴⁾ These findings suggest that the PGE₂-EP4 pathway in adipocytes is a determinant of whether obese people acquire metabolically obese or healthy characters.

Fig. 2. PGE₂-EP4 Pathway Regulates Physiological Lipolysis upon Insulin Stimulation

PGE₂ production is induced after feeding by the action of ATGL, COX-1, and mPGES1 in adipocytes. PGE₂ promotes basal lipolysis and lipid transport into other tissues such as the liver via the EP4 receptor. DAG, diacylglycerol; MAG, monoacylglycerol, G0S2, G0/G1 switch gene 2.

In the morbidly obese state, inflammatory cytokines, such as interleukin-6 and TNF-α, or chemokines, including monocyte chemoattractant protein-1 and regulated on activation, normal T cell expressed and secreted (RANTES) are released from adipocytes, resulting in chronic inflammation in WAT.^45–48) It was shown that COX-2 expression is enhanced in the adipocytes of obese rodents and humans, and that EP3 and EP4 have reciprocal actions to each other in the control of chronic inflammation, as observed in the regulation of lipolysis in WAT.⁴⁹⁾ The treatment of DIO model mice with a COX-2 inhibitor or an EP3 antagonist results in the downregulation of proinflammatory adipokines. In contrast, an EP4 agonist was shown to suppress WAT inflammation.^50,51) EP3 and EP4 receptors, which are coupled to the Gi and Gs protein, respectively, compete with each other both in intracellular signaling and inflammatory regulation, and hence the balance of the signaling of these two receptors appears to determine the severity of chronic inflammation in WAT. Interestingly, the expression levels of EP3 and EP4 in adipocytes was shown to be increased and decreased, respectively in both obese rodents and diabetic patients.⁴⁹⁾ Therefore, the contribution of the ‘harmful’ PGE₂-EP3 pathway may become more dominant along with the progression of obesity.

5. ROLES OF PROSTANOID RECEPTORS IN THE REGULATION OF ADIPOCYTE BROWNING

Adipose tissue browning is induced by several stimuli, such as cold stress, and it has long been thought that the β₃ receptor, which is an adipocyte-specific β-adrenergic receptor, performs a central role in the adipocyte browning process in rodents.^52,53) β₃ agonists were therefore expected to be promising drugs for the treatment of metabolic disorders, and pharmaceutical companies have tried to develop a number of compounds for clinical use. However, clinical trials for these drugs have been unsuccessful to date because of the low efficiency or selectivity of these drugs.⁵⁴⁾ Moreover, it was recently reported that β₃ receptor-deficient mice with specific genetic backgrounds show a normal response in cold-induced browning and thermogenesis, and from these findings, the existence of an alternative β₃ receptor-independent pathway that regulates the browning process was elucidated.⁵⁵⁾

In 2010, two groups simultaneously demonstrated that the browning process is mediated by COX-2-derived prostanoids. Vegiopoulus et al. reported that browning was induced in cutaneous-specific COX-2 transgenic mice, and that COX-2 inhibitor treatment blocked β3 agonist-induced browning.⁵⁶⁾ Several years later, this group showed that a stable analog of PGI₂ induced the conversion of white to brite adipocytes via the IP-cAMP pathway and the peroxisome proliferator-activated receptor gamma (PPARγ)-dependent pathway.^57,58) On the other hand, Madsen et al. found that COX-2-deficient mice demonstrate a diminished browning response and a lower ability to maintain body temperature against cold stress than wild-type mice.⁵⁹⁾ They further showed the possibility that the PGE₂-EP4 pathway mediates the induction of browning, using MEFs. In contrast to these findings, Paschos et al. reported that the cold-induced browning response is intact in two types of conditional COX-2-deficient mice, namely, postnatal global knockout or adipocyte-specific knockout lines.⁶⁰⁾ Therefore, the exact contribution of COX-2 to cold-induced browning remains to be determined. Recently, a novel regulatory mechanism has been proposed for the synthesis of prostanoids in the induction of adipocyte browning. Zhang et al. demonstrated that mechanistic target of rapamycin complex 1 (mTORC1), a molecule activated by nutrient stress such as a high-fat diet, suppresses COX-2-dependent browning, using mice deficient in rapamycin-associated TOR protein (Raptor), which is a key component of mTORC1.⁶¹⁾ Activated mTORC1 phosphorylates cAMP response element binding protein (CREB)-regulated transcription coactivator 2 (CRTC2), and subsequently dissociates CREB from the COX-2 promoter, resulting in COX-2 downregulation in adipocytes (Fig. 3). Sharmsi et al. reported that cold stress- or exercise-induced fibroblast growth factor (FGF) 6 or FGF9 expression in WAT induces browning via the FGFR3 receptor in a COX-2/mPGES1/PGE₂-dependent manner.⁶²⁾ Moreover, they found that the PGE₂-EP2/EP4 pathway enhances estrogen receptor-related alpha (ERRα) expression, and its recruitment to the intranuclear UCP1 enhancer region, resulting in UCP1 upregulation in adipocytes (Fig. 3). Another group also proposed that the FGF8b-FGFR1 pathway induces adipocyte UCP1 expression by COX-2/mPGES1-dependent PGE₂ synthesis.⁶³⁾ From these studies using rodents, EP2, EP4, and IP, which are all Gs-coupled GPCRs, can be considered as potential browning inducers. However, it remains to be elucidated as to which receptor mainly contributes to physiological browning, and whether the browning process is mediated by prostanoids also in humans, towards the future clinical application of these receptor-targeting drugs.

Fig. 3. Deduced Roles of Prostanoids in Adipocyte Browning

mTORC1 activation by nutrient stress suppresses, whereas cold or exercise-induced FGF receptor signaling enhances COX-2 expression in adipocytes. PGE₂ and PGI₂ produced by COX-2 promotes brown adipogenesis from progenitor cells by UCP1 upregulation via the EP2/EP4 and IP receptor, respectively.

6. CONCLUDING REMARKS

As mentioned above, prostanoids have recently been identified as a crucial regulator of adipocyte function, and they have therefore gained attention as therapeutic targets of metabolic disorders (Table 1). However, the clinical application of compounds targeting prostanoid signaling for metabolic modulation has not been accomplished. This is probably because various prostanoid receptors positively and negatively regulate each metabolic process in a cell type- or disease background-dependent manner. Recent studies have demonstrated the molecular mechanisms underlying the metabolic regulation by each prostanoid receptor. Application of this knowledge is expected to lead to the development of prostanoid receptor-specific or multiple PG receptor-targeting drugs, and furthermore, the design of methods to deliver these drugs to specific tissues is expected to lead to the establishment of more effective therapies for metabolic disorders in the future.

Table 1. Summary of the Biological Actions of Prostanoid Receptor Pathways in Adipose Tissues Cited in This Review

Ligand	Receptor	Biological action	References
PGE2	EP2	Promotion of adipose tissue browning	62)
	EP3	Suppression of adipogenesis	38)
		Suppression of lipolysis	39,40)
		Promotion of WAT inflammation	49)
	EP4	Suppression of adipogenesis	36,37)
		Promotion of lipolysis	43)
		Suppression of WAT inflammation	50,51)
		Promotion of adipose tissue browning	59,62)
PGF2α	FP	Suppression of adipogenesis	32–34)
PGI2	IP	Promotion of adipogenesis	28–31)
		Promotion of adipose tissue browning	57,58)

Conflict of Interest

The authors declare no conflict of interest.

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