The EP3 receptor/G_z signaling axis as a therapeutic target for diabetes and cardiovascular disease

Abstract

Cardiovascular disease is a common co-morbidity found with obesity-linked type 2 diabetes. Current pharmaceuticals for these two diseases treat each of them separately. Yet, diabetes and cardiovascular disease share molecular signaling pathways that are increasingly being understood to contribute to disease pathophysiology, particularly in pre-clinical models. This review will focus on one such signaling pathway: that mediated by the G protein-coupled receptor, Prostaglandin E₂ Receptor 3 (EP3), and its associated G protein in the insulin-secreting beta-cell and potentially the platelet, G_z. The EP3/G_z signaling axis may hold promise as a dual target for type 2 diabetes and cardiovascular disease.

Introduction

Diabetes is a chronic and life-threatening disease whose overall life risk has risen dramatically over the last two decades. In 2014, the Centers for Disease Control reported more than 29 million people in the US (9.3% of the population) with diagnosed or undiagnosed diabetes: primarily obesity-linked type 2 diabetes (T2D)(1). Further, in this same time period 86 million American adults were estimated to have pre-diabetes, and although it is traditionally considered adult onset, T2D affected the lives of thousands of children and adolescents (1). In addition to the indirect impact of T2D on productivity and healthy lifespan, the direct financial impact in the US is nearly $250 billion healthcare dollars per year, and rising (2).

T2D is tightly correlated with obesity and is a metabolic disease in which tissues normally sensitive to the peptide hormone, insulin, become resistant to its action. This, in conjunction with dysfunction and/or death of the insulin-secreting pancreatic β-cells, ultimately leads to a failure to maintain healthy blood glucose levels (3, 4). The loss of insulin secretion and function results in a host of metabolic defects, including hyperglycemia, and is associated with chronic comorbidities including hyperlipidemia, retinopathy, and cardiovascular disease (3).

Poorly managed T2D increases the risk for complications and comorbid conditions that adversely affect quality of life (3, 5). The vast increase in cardiovascular death rates and incidences of myocardial infarction and ischemic stroke by adults diagnosed with diabetes is alarming as compared to non-diabetics (6). The pathophysiology of cardiovascular disease and diabetes are tightly linked through many complex factors, including inflammation (6). Despite recent advancements in pharmaceutical therapies, treatments for T2D often fail, or are insufficient in managing blood glucose homeostasis (7) and preventing the associated disease risks.

There is an impending need for improved T2D therapies strategically targeting T2D pathology and its associated co-morbidities such as cardiovascular disease. Currently, a significant proportion of all pharmaceuticals target a specific membrane receptor family, the G protein-coupled receptors (GPCRs) (8). Many GPCRs are involved in pathways that could ultimately improve target tissue insulin sensitivity, β-cell function, and health (9). The most well-known GPCR as a T2D therapeutic target is the glucagon-like peptide 1 receptor (GLP-1R), a stimulatory G_s-coupled receptor that acts in multiple tissues to decrease the gastric emptying rate, improve insulin action, and improve insulin secretion (9, 10). In addition, recent studies have suggested a lower risk of cardiovascular events in individuals treated with certain GLP-1-based therapeutics (11–13). Yet, GLP-1-based therapeutics are not effective to reduce blood glucose in all individuals with diabetes (14). It is now becoming clear that inhibitory G_i-coupled receptors serve a counter-regulatory role in the beta-cell, and that these GPCR pathways can be up-regulated in T2D (15–19). In this review, we will give an overview of GPCRs as T2D therapeutic targets as well as discuss how a specific receptor, Prostaglandin E2 Receptor 3 (EP3), and its beta-cell-associated G-protein, G_z, have the potential to act as therapeutic targets for T2D and cardiovascular disease.

GPCRs as Therapeutic Targets for T2D

Traditional T2D therapies have focused on directly increasing peripheral insulin sensitivity and/or activating insulin secretory pathways in the β-cell. Metformin and other insulin sensitizers target peripheral and hepatic tissues to restore insulin sensitivity, thereby reducing blood glucose by reducing hepatic glucose output and improving peripheral glucose uptake. Metformin is the classical first-line therapeutic for T2D and has been in use since the 1950s, establishing itself with an excellent cardiovascular risk profile (20). Yet, many individuals progress to requiring additional pharmaceutical treatment in order to maintain healthy blood glucose levels. The thiazolidinedione, rosiglitazone, is a potent insulin sensitizer, but is associated with an increased risk of myocardial infarction, and is currently under selling restrictions in the US (20). Sulfonylureas and meglintides act on the ATP-sensitive potassium (K_ATP) channel to stimulate beta-cell membrane depolarization and exocytosis of insulin granules (21, 22), and are often combined with metformin as a dual therapy (23). Yet, the cardiovascular risk of sulfonylureas is questionable(20), and meglintides have not been studied in enough detail to determine their cardiovascular safety (20).

The GLP-1R was originally proposed as a T2D therapeutic due to its ability to promote insulin secretion from the beta-cell in a glucose-dependent manner (24), in contrast to sulfonylureas and meglintides, whose effects on insulin secretion are fully or partially glucose-independent, respectively. GLP-1 is now known to have effects on many other target tissues, acting in an autocrine, paracrine, or neuronal manner due to its rapid degradation in circulation by the activity of dipeptidyl peptidase 4 (DPP4) (25). Stable GLP-1 analogs such as exenatide and liraglutide and DPP4 inhibitors such as sitagliptin and vildagliptin have been in use clinically since the mid-late 2000s and are currently well-accepted therapeutics for T2D due to their positive effects on insulin secretion and whole-body glucose homeostasis. Further, while the cardiovascular risks of DPP-4 inhibitors is still unclear, the GLP-1 analogs have a very favorable cardiovascular risk profile (20). Yet, GLP-1-based therapeutics do not work to effectively reduce blood glucose in all individuals. For example, approximately 35–60% of T2D patients treated with sitagliptin fail to achieve their target of glycosylated hemoglobin levels (HbA1c) of less than 7 percent (26–28).

Most of what we know about GLP-1R signaling and therapeutic effects has been derived from rodent studies. In the beta-cell, ligand binding to the GLP-1R potentiates glucose stimulated insulin secretion through elevations in intracellular cyclic AMP (cAMP) production to induce the activity of protein kinase A (PKA) and EPAC2 (a guanine nucleotide exchange factor for Rap1 directly activated by cAMP), leading to increased glucose metabolism, membrane depolarization, Ca²⁺ influx, Ca²⁺-induced Ca²⁺ release (CICR) and direct activation of the exocytotic machinery on insulin secretory granules (Figure 1)(29–33). While activation of the Gs protein can stimulate cAMP production, in the beta-cell, effects on cAMP signaling and insulin secretion require beta-arrestin-1 (34). GLP-1R activation also induces β-cell hypertrophy and proliferative gene expression through the cAMP regulatory element binding protein (CREB) (Figure 1) (29, 30). Moreover, GLP-1 has effects in the hypothalamus that suppress appetite and increase energy expenditure, important aspects to controlling T2D pathophysiology. For further reading on the systemic effects of GLP-1, we direct readers to the comprehensive review by Baggio and Drucker (24).

Figure 1: Modulation of beta cell function and survival by prostaglandins.

Glucose enters beta cells via GLUT2 and then is metabolized, increasing the ATP/ADP ratio. K_ATP channels close due to the abundance of ATP, promoting membrane depolarization and the opening of voltage-dependent calcium channels (VDCCs) and influx of calcium. Calcium induces further intracellular calcium release and, along with ATP, stimulates exocytosis of insulin granules. Glucose-stimulated insulin secretion as well as proliferation and survival can be modulated by cyclic AMP (cAMP). Glucagon-like peptide-1 (GLP-1) enhances beta-cell function, proliferation and survival via activation of its G protein-coupled receptor (GPCR), GLP1R, which is coupled to a stimulatory G protein that activates adenylate cyclase to generate cAMP. Beta-cell cAMP signaling and effects on insulin secretion require the presence of beta-arrestin-1 (β-arr-1). Conversely, the arachidonic acid metabolite, prostaglandin E₂ (PGE₂) activates a different GPCR, EP3, to inhibit cAMP production and its downstream effects. Intracellular cAMP works by multiple mechanisms to regulate glucose-stimulated insulin secretion and proliferation and survival. Stimulation of EPAC2 by cAMP promotes release of GDP from Rap1 and binding of the more cellular abundant GTP resulting in Rap1’s active conformation and enhanced glucose stimulated insulin secretion. Rap1 activity is limited by Rap1GAP, which is a unique effector of G_z. PKA phosphorylates many substrates to enhance calcium mobilization and nuclear translocation of cAMP bound CREB to promote proliferation and survival.

Chronic inflammation, obesity and T2D pathogenesis

Metabolic tissues maintain immune homeostasis, and it is well established that obesity leads to chronic low-grade inflammation (35, 36). Immune infiltration in adipose, hepatic and pancreatic islet tissues has been well documented (37–39). Elevated macrophage infiltration into tissues leads to a pro-inflammatory cytokine profile, including changes in TNFα, IL-6, and IL-1β (40–42). Pro-inflammatory cytokine effects on metabolic tissues have been characterized mechanistically, and, in general, lead to alterations in the insulin receptor signaling pathway that induce insulin resistance. The induction of insulin resistance impairs glucose transport in insulin-sensitive tissues, leading to hyperglycemia (43–45). In this state, hepatic tissues are incapable of properly managing glucose homeostasis through insulin regulatory mechanisms that halt gluconeogenesis and induce glycogen storage, exacerbating hyperglycemia (46, 47).

Pro-inflammatory cytokines also induce prostaglandin (PG) synthesis, and disrupted PG profiles due to elevated pro-inflammatory signals have been documented in the obese, insulin resistant, and diabetic states (44, 48–54). PGs are transient lipid-derived molecules possessing a wide variety of effects on various tissues. Pro-inflammatory signal transduction activates the lipolytic enzyme, phospholipase A2, which functions to cleave AA from the membrane embedded phospholipids. Free AA is synthesized to prostaglandin G₂ and prostaglandin H₂ through a series of oxygenation and reduction reactions by the cyclooxygenase enzymes COX-1 and COX-2. PGH₂ is the central precursor for all PGs. Its subsequent metabolism is tissue specific, but prostaglandin synthase enzymes result in the production of PGD₂, PGF_2α, PGE₂, prostacyclin (PGI₂), and thromboxane A2 (TXA₂). Of primary concern in T2D is the negative effect of PGs on pancreatic islets as glucose homeostasis cannot be restored or managed without exogenous insulin once functional beta-cell mass has significantly declined.

Prostaglandin E₂, the EP3 receptor and diabetes pathogenesis.

PGE₂ and its receptor, Prostaglandin E₂ receptor 3 (EP3), have been proposed as contributors to the progression of obesity, insulin resistance, and β-cell failure (48, 54, 55). PGE₂ is the primary endogenous ligand for the E prostanoid GPCR family: EP1, EP2, EP3 and EP4. EP1 is a G_q-coupled GPCR whose activation induces phospholipase C beta (PLC-β) activity, triggering cytoplasmic Ca²⁺ influx. EP2 and EP4 are both Gs-coupled GPCRs responsible for elevated adenylate cyclase activity and cAMP production, as well as Ca²⁺ influx. Of the four receptors, only EP3 couples to G_i proteins, making it the only PGE₂ receptor that reduces cAMP production upon ligand binding, while also inhibiting cytoplasmic Ca²⁺ flux via reduction in PKA, EPAC, and G_βγ activity.

The first elucidation of the G protein coupling and biological activity of EP3 in the β-cell was by the Robertson group using the hamster insulinoma cell line, HIT-T15. PGE₂ binds specifically to HIT-T15 cell membranes and elicits a dose-dependent decrease in cAMP accumulation and insulin secretion (17). PGE₂ binds in a similar manner to isolated guinea pig islets (17). In the presence of pertussis toxin (PTX)—which inactivates nearly all Gα_i subfamily members by catalyzing the ADP ribosylation of a cysteine near their C termini—the maximal inhibitory effect of PGE₂ on cumulative insulin secretion is reduced but not ablated, suggesting coupling of the HIT-T15 EP3 receptor to both PTX-sensitive and -insensitive G_i proteins (17). Perifusion assays to measure temporal insulin secretion in HIT-T15 cells demonstrate a single phase of insulin secretion when stimulated by glucose, with the addition of the inhibitor of cAMP degradation, isobutyl-1-methylxanthine (IBMX), providing a second phase of insulin secretion more consistent with that observed from isolated pancreatic islets (18). Addition of PGE₂ to the perifused solution reduced insulin secretion by approximately 50% in both cases, while PTX pre-treatment reduced but did not completely block this inhibition (18). Interestingly, continuing the PTX treatment during the assay almost completely ablated the effects of PGE₂ on inhibiting insulin secretion, although this was not necessarily explained by a lack of ADP ribosylation of G_i proteins, as both pre-treatment and continuous treatment of cells with PTX displayed the same completeness of ADP-ribosylation of Gα subunits (18). Further work by the Robertson group revealed the identity of PTX-sensitive G_i/o subfamily members in HIT-T15 cells: Gα_i1, Gα_i2, Gα_i3, and Gα_o subtypes including Gα_o2 (56).

Because of the partial PTX insensitivity of the inhibitory response of PGE₂ in HIT-T15 cells, we were intrigued with the possibility of EP3 coupling to the only PTX-insensitive G_i subfamily member, G_z (57). Gα_z protein is readily detectable in isolated rat and mouse islets and insulinoma cell lines by Western blot or immunohistochemistry (58). Using the Ins-1(832/13) rat insulinoma cell line, the closely related eicosanoid, PGE₁ (which has nearly the same affinity profile for the mouse EP receptors as PGE₂ (59)) inhibited insulin secretion by approximately 50%, an effect that was unchanged by PTX pre-treatment (58). Overexpression of the RGS domain of the specific Gα_z inhibitor, RGSZ1, or siRNA-mediated reduction in Gα_z protein expression, completely or partially ablated the effect of PGE₁ on insulin secretion, respectively, supporting the coupling of EP3 to G_z (58). The PTX-insensitivity of EP3 activity was confirmed in islets isolated from diabetic C57BL/6J Leptin^Ob/Ob mice, which show a 50% inhibition of insulin secretion by the selective EP3 agonist, sulprostone, an effect that is unchanged by PTX pre-treatment (60). In both the 2005 and 2012 publications cited above, the inhibition of insulin secretion by other inhibitory GPCR agonists, including those targeting the α2A adrenergic receptor and the somatostatin receptor, were completely ablated by PTX, demonstrating a full inhibition of PTX-sensitive G_i proteins (58, 60). Taken together, the studies by the Robertson and Kimple groups are not necessarily discordant, and differences in the ability of PGE₁, PGE₂, or sulprostone to activate G_i/o vs. G_z proteins; the concentration of PTX used; and/or species-specific differences in GPCR coupling or G protein expression could explain the partial vs. full PTX insensitivity of EP3 signaling found by the two groups. In sum, while EP3 may couple to other G_i proteins, it is certainly at least partially coupled to G_z in both insulinoma cell lines and isolated pancreatic islets (shown in Figure 1).

Elevated PGE₂ acts in an autocrine or paracrine manner to bind to EP3. PGE₂ is released into the media from cultured islet cells or insulinoma cell lines incubated in stimulatory glucose, effects that are completely ablated by addition of COX inhibitors (17, 61). Elevated PGE₂ production is also observed from cultured mouse or human islets isolated from diabetic individuals (54). Coincidently, EP3 expression itself is also induced in islets isolated from diabetic mice and humans and corresponds with reduction in cAMP signaling and a blunted GLP-1 response in the β-cell (54).

The EP3/G_z signaling pathway has been proposed as a therapeutic target for beta-cell failure in T2D (54, 60). C57BL/6N mice lacking the catalytic alpha-subunit of G_z (Gα_z-null) are resistant to glucose intolerance induced by high-fat feeding as a result of increased β-cell replication, leading to elevated insulin secretion even in the face of severe insulin resistance (60). This same mouse model, when administered a low daily dose of exenatide, is resistant to hyperglycemia induced by the beta-cell toxin streptozotocin due to improved beta-cell replication, survival, and function (62). These results lend strong support to the EP3/G_z signaling pathway as a therapeutic target for T2D. Yet, nearly all pharmaceuticals that target G-protein signaling pathways target the GPCR and not the G protein or other downstream signaling components, and EP3-null mice have been shown to have increased daytime food intake as compared to wild-type controls, which, over time, leads to increased adiposity and body weight (63). These mice exhibit fasting hyperglycemia, increased plasma insulin and leptin levels, and worsened insulin sensitivity and glucose tolerance as compared to wild-type controls at both 3 and 6 months of age (63). These metabolic findings were re-capitulated in a separate study, which also found leaving the EP3β variant intact was insufficient to protect from the spontaneous development of obesity, insulin resistance, and fasting hyperinsulinemia (55). Finally, a third study with whole-body EP3-null mice found that chow-fed animals have an essentially wild-type metabolic phenotype, while high-fat diet-fed EP3-null mice exhibit increased food intake, increased adiposity, more severe insulin resistance, and elevated serum triglycerides as compared to wild-type mice (48). This leads high-fat diet-fed EP3-null mice to have fasting hyperglycemia and fasting hyperinsulinemia with more severe glucose intolerance than wild-type controls, hallmarks of the diabetic condition (48).

Ceddia and colleagues determined there is no effect of EP3 loss on beta-cell function when pancreatic islets were assayed ex vivo (48). Therefore, the phenotype of the EP3-null mouse is likely not a beta-cell phenotype. EP3 is the E prostanoid receptor variant most highly expressed in white adipose tissue (64). The adipose tissue expression of total EP3 or any of its three splice variants is significantly down-regulated in mouse models of diabetes (55), and specific activation of adipose-tissue EP3 increases adiposity and decreases lipolysis (65). These results are consistent with the phenotype of EP3-null mice, which have increased adipogenesis and adipocyte size, contributors to the obesity phenotype, and augmented lipolysis (48, 55). Furthermore, the adipose tissue itself is more necrotic and inflamed in high-fat diet-fed EP3-null mice, leading to increased secretion of inflammation-associated adipokines (48). Finally, inhibition of adipocyte COX-2 augments obesity-associated adipose tissue inflammation and insulin resistance (50). Taken together, these results suggest systemic inhibition of EP3 may not be an appropriate T2D therapeutic strategy, but instead support a beta-cell-specific or biased antagonist therapy that would directly interfere with up-regulated EP3/G_z signaling in the beta-cell, leaving the beneficial effects of EP3 in tissues such as adipose intact.

Role of G_i proteins in cardiovascular disease

Platelets are key contributors to the development of cardiovascular disease, a primary co-morbid condition occurring in T2D. Like pancreatic β-cells, platelet function can be regulated by inhibitory G protein signaling. Platelets express four Gα_i family members: Gα_i1, Gα_i2, Gα_i3 and Gα_z (66–68). The preferential coupling of Gα_i family members to GPCRs was demonstrated using specific Gα subunit knock-out mice. Gα_i2 preferentially couples to the ADP receptor P2Y12 (69) and Gα_z prefers the α2A-adrenergic receptor for epinephrine (Figure 2) (70). Gα_i2 and Gα_z, but not Gα_i3, regulate basal levels of cAMP in circulating platelets (71).

Figure 2: Regulation of platelet function by prostanoid receptors.

Prostaglandins, particularly prostaglandin E₂ (PGE₂), can have a profound impact on platelet function based on the prostanoid receptor that becomes activated. IP and EP4 couple to stimulatory heterotrimeric G proteins to activate adenylate cyclase and enhance cAMP production. In direct contrast, EP3 couples to an inhibitory G protein to prevent cAMP generation. Intracellular cAMP levels and activation of the PKA negatively influences platelet reactivity by phosphorylation and inactivation of specific proteins important to many aspects of platelet activation. Granule release and integrin αIIbβ3 activation can be prevented by PKA phosphorylation of the IP3 receptor (IP3R) on the ER, which prevents IP3-stimulated release of calcium and subsequent activation of the small GTPase Rap1 via CalDAG GEF1. PKA phosphorylation of CalDAG GEF1 can also impair its ability to activate Rap1. In contrast, βγ subunits previously associated with inhibitory G proteins have been reported to activate PI3K to inhibit RASA3 to promote prolonged activation of Rap1, thereby stimulating granule release and integrin activation.

In addition to the classical role of Gα_i proteins in the inhibition of AC activity and reduction of cAMP, the Gβ_γ subunits of G proteins activate phosphoinositide 3-kinase (PI3K) to elicit signaling to promote platelet activation (66). Recent studies indicate that PI3K inhibits Rasa3, a Rapl GTPase activating protein, to promote platelet activation (Figure 2) (72).

It has been long appreciated that diabetics have increased sensitivity of platelets to ADP and epinephrine, two agents well-known to stimulate platelet activation (73). In both pre- clinical models and diabetic subjects, elevated plasma levels of the PGE2 metabolite, 15- keto-13,14-dihydroprostaglandin E2, can be found (60, 74–76). This is particularly evident during diabetic ketoacidosis (75, 76). Isolated whole blood or platelet-rich plasma from type 1 and/or type 2 diabetic subjects produce significantly more prostaglandin E-like material or PGE2 itself than isolates from control subjects (77, 78). In addition, platelets isolated from diabetic subjects generate increased amounts of prostaglandin E-like material or PGE2 itself in response to ADP, epinephrine, collagen and arachidonic acid (73, 79).

PGE₂ can have profound effects on platelet function by binding to and activating EP receptors. The non-steroidal anti-inflammatory drugs (NSAIDs), such as acetylsalicylic acid (aspirin) target the COX enzymes to inhibit prostaglandin production, abolishing platelet hypersensitivity. Using receptor subtype specific or selective agonists and E prostanoid receptor-null mice, it was demonstrated that EP3, EP4 and IP receptors elicit functional responses in platelets. EP4 and IP receptors inhibited platelet function and enhanced VASP phosphorylation (80, 81), indicating that these two GPCRs are G_s coupled. Conversely, the EP3 receptor agonist promotes platelet aggregation, calcium signaling and P-selectin expression, which are associated with reduction of VASP phosphorylation (80, 81), suggesting coupling to a G_i family member. Antagonist-induced competitive inhibition of EP3 decreases thrombosis without impairing hemostasis (82).

EP3 antagonists have been suggested as anti-platelet activating therapeutics. Due to EP3 and G_z coupling in pancreatic β-cells (54), it is possible that EP3 also couples to G_z in platelets. Unfortunately, this functional relationship has not yet been confirmed, but is suggested in Figure 3: a summary of EP3 functions in various insulin-sensitive tissues and platelets and how these may be impacted in the diabetic environment.

Figure 3: PGE₂ signaling contributes to the diabetic environment.

Chronic low-grade inflammation and hyperglycemia are hallmarks of diabetes mellitus with elevated circulating levels of pro-inflammatory cytokines, TNFα, IL-6 and IL-1β. These cytokines alter insulin receptor signaling leading to insulin resistance and also to enhanced synthesis of prostaglandins, including PGE₂, in target tissues. PGE₂ is rapidly degraded in the circulation, but elevated plasma levels of the PGE₂ metabolite, 15-keto-13,14-dihydroprostaglandin E₂ (dhPGE₂), have been found in pre-clinical models of diabetes as well as individuals with diabetes mellitus. EP3-expressing, insulin-sensitive tissues such as adipose and liver become insulin resistant and contribute to prolonged hyperglycemia in different ways. Insulin resistance in hepatic tissues enhances gluconeogenesis and inhibits storage of glycogen, while adipocytes fail to uptake glucose for energy use. In addition, EP3 is pathologically up-regulated with diabetes onset in pancreatic islets. The coupling of EP3 to the inhibitory heterotrimeric G protein, G_z, in pancreatic islets results in reduced glucose stimulated insulin secretion and impaired maintenance of beta cell mass. Due to endothelial cell activation in the diabetic environment, the resting tone of platelets is perturbed leading to hypersensitivity. Independently and/or possibly together, platelet EP3 and G_z contribute to signaling pathways leading to platelet activation.

Targeting EP3 and Ga _zln Diabetes

Prostaglandins are of great physiological importance and are required for proper immune function, among other physiological processes. Furthermore, while plasma levels of the PGE₂ metabolite, 15-keto-13,14-dihydroprostaglandin E₂, are elevated in the diabetic condition, 15-keto-13,14-dihydroprostaglandin E₂ itself does not activate the EP3 receptor and its utility as a biomarker for either disease status or therapeutic response remains questionable. Finally, while EP3 receptor signaling is dyfunctionally up-regulated in the diabetic beta-cell, its expression in other tissues is required for normal processes such as the inhibition of lipolysis. Taken together, these findings suggest systemic targeting of prostaglandin production or direct inhibition of EP3 may not be successful pharmacological strategies for treating diabetes and its complications. However, the function of EP3 and its putative coupling to G_z, whose expression is quite limited, in both pancreatic islets and platelets provides a unique scenario in which development of biased- antagonist specifically targeting EP3 and G_z coupling could prove useful in the direct treatment of both hyperglycemia as well as associated cardiovascular disease.