Use of DREADD Technology to Identify Novel Targets for Antidiabetic Drugs.

G protein-coupled receptors (GPCRs) form a superfamily of plasma membrane receptors that couple to four major families of heterotrimeric G proteins, Gs, Gi, Gq, and G12. GPCRs represent excellent targets for drug therapy. Since the individual GPCRs are expressed by many different cell types, the in vivo metabolic roles of a specific GPCR expressed by a distinct cell type are not well understood. The development of designer GPCRs known as DREADDs (designer receptors exclusively activated by a designer drug) that selectively couple to distinct classes of heterotrimeric G proteins has greatly facilitated studies in this area. This review focuses on the use of DREADD technology to explore the physiological and pathophysiological roles of distinct GPCR/G protein cascades in several metabolically important cell types. The novel insights gained from these studies should stimulate the development of GPCR-based treatments for major metabolic diseases such as type 2 diabetes and obesity.

Metabolic effects of skeletal muscle-specific deletion of beta-arrestin-1 and -2 in mice.

Type 2 diabetes (T2D) has become a major health problem worldwide. Skeletal muscle (SKM) is the key tissue for whole-body glucose disposal and utilization. New drugs aimed at improving insulin sensitivity of SKM would greatly expand available therapeutic options. ß-arrestin-1 and -2 (Barr1 and Barr2, respectively) are two intracellular proteins best known for their ability to mediate the desensitization and internalization of G protein-coupled receptors (GPCRs). Recent studies suggest that Barr1 and Barr2 regulate several important metabolic functions including insulin release and hepatic glucose production. Since SKM expresses many GPCRs, including the metabolically important ß2-adrenergic receptor, the goal of this study was to examine the potential roles of Barr1 and Barr2 in regulating SKM and whole-body glucose metabolism. Using SKM-specific knockout (KO) mouse lines, we showed that the loss of SKM Barr2, but not of SKM Barr1, resulted in mild improvements in glucose tolerance in diet-induced obese mice. SKM-specific Barr1- and Barr2-KO mice did not show any significant differences in exercise performance. However, lack of SKM Barr2 led to increased glycogen breakdown following a treadmill exercise challenge. Interestingly, mice that lacked both Barr1 and Barr2 in SKM showed no significant metabolic phenotypes. Thus, somewhat surprisingly, our data indicate that SKM ß-arrestins play only rather subtle roles (SKM Barr2) in regulating whole-body glucose homeostasis and SKM insulin sensitivity.

Oxidative stress modulates nucleobase transport in microvascular endothelial cells.

Purine nucleosides and nucleobases play key roles in the physiological response to vascular ischemia/reperfusion events. The intra- and extracellular concentrations of these compounds are controlled, in part, by equilibrative nucleoside transporter subtype 1 (ENT1; SLC29A1) and by equilibrative nucleobase transporter subtype 1 (ENBT1). These transporters are expressed at the membranes of numerous cell types including microvascular endothelial cells. We studied the impact of reactive oxygen species on the function of ENT1 and ENBT1 in primary (CMVEC) and immortalized (HMEC-1) human microvascular endothelial cells. Both cell types displayed similar transporter expression profiles, with the majority (>90%) of 2-chloro[(3)H]adenosine (nucleoside) uptake mediated by ENT1 and [(3)H]hypoxanthine (nucleobase) uptake mediated by ENBT1. An in vitro mineral oil-overlay model of ischemia/reperfusion had no effect on ENT1 function, but significantly reduced ENBT1 Vmax in both cell types. This decrease in transport function was mimicked by the intracellular superoxide generator menadione and could be reversed by the superoxide dismutase mimetic MnTMPyP. In contrast, neither the extracellular peroxide donor TBHP nor the extracellular peroxynitrite donor 3-morpholinosydnonimine (SIN-1) affected ENBT1-mediated [(3)H]hypoxanthine uptake. SIN-1 did, however, enhance ENT1-mediated 2-chloro[(3)H]adenosine uptake. Our data establish HMEC-1 as an appropriate model for study of purine transport in CMVEC. Additionally, these data suggest that the generation of intracellular superoxide in ischemia/reperfusion leads to the down-regulation of ENBT1 function. Modification of purine transport by oxidant stress may contribute to ischemia/reperfusion induced vascular damage and should be considered in the development of therapeutic strategies.

In vivo metabolic effects after acute activation of skeletal muscle Gs signaling.

OBJECTIVE: The goal of this study was to determine the glucometabolic effects of acute activation of Gs signaling in skeletal muscle (SKM) in vivo and its contribution to whole-body glucose homeostasis. METHODS: To address this question, we studied mice that express a Gs-coupled designer G protein-coupled receptor (Gs-DREADD or GsD) selectively in skeletal muscle. We also identified two Gs-coupled GPCRs that are endogenously expressed by SKM at relatively high levels (ß2-adrenergic receptor and CRF2 receptor) and studied the acute metabolic effects of activating these receptors in vivo by highly selective agonists (clenbuterol and urocortin 2 (UCN2), respectively). RESULTS: Acute stimulation of GsD signaling in SKM impaired glucose tolerance in lean and obese mice by decreasing glucose uptake selectively into SKM. The acute metabolic effects following agonist activation of ß2-adrenergic and, potentially, CRF2 receptors appear primarily mediated by altered insulin release. Clenbuterol injection improved glucose tolerance by increasing insulin secretion in lean mice. In SKM, clenbuterol stimulated glycogen breakdown. UCN2 injection resulted in decreased glucose tolerance associated with lower plasma insulin levels. The acute metabolic effects of UCN2 were not mediated by SKM Gs signaling. CONCLUSIONS: Selective activation of Gs signaling in SKM causes an acute increase in blood glucose levels. However, acute in vivo stimulation of endogenous Gs-coupled receptors enriched in SKM has only a limited impact on whole-body glucose homeostasis, most likely due to the fact that these receptors are also expressed by pancreatic islets where they modulate insulin release.

Clenbuterol exerts antidiabetic activity through metabolic reprogramming of skeletal muscle cells.

Activation of the sympathetic nervous system causes pronounced metabolic changes that are mediated by multiple adrenergic receptor subtypes. Systemic treatment with ß2-adrenergic receptor agonists results in multiple beneficial metabolic effects, including improved glucose homeostasis. To elucidate the underlying cellular and molecular mechanisms, we chronically treated wild-type mice and several newly developed mutant mouse strains with clenbuterol, a selective ß2-adrenergic receptor agonist. Clenbuterol administration caused pronounced improvements in glucose homeostasis and prevented the metabolic deficits in mouse models of ß-cell dysfunction and insulin resistance. Studies with skeletal muscle-specific mutant mice demonstrated that these metabolic improvements required activation of skeletal muscle ß2-adrenergic receptors and the stimulatory G protein, Gs. Unbiased transcriptomic and metabolomic analyses showed that chronic ß2-adrenergic receptor stimulation caused metabolic reprogramming of skeletal muscle characterized by enhanced glucose utilization. These findings strongly suggest that agents targeting skeletal muscle metabolism by modulating ß2-adrenergic receptor-dependent signaling pathways may prove beneficial as antidiabetic drugs.

Nucleoside/nucleobase transport and metabolism by microvascular endothelial cells isolated from ENT1-/- mice.

Nucleoside and nucleobase uptake is integral to mammalian cell function, and its disruption has significant effects on the cardiovasculature. The predominant transporters in this regard are the equilibrative nucleoside transporter subtypes 1 (ENT1) and 2 (ENT2). To examine the role of ENT1 in more detail, we have assessed the mechanisms by which microvascular endothelial cells (MVECs) from ENT1(-/-) mice transport and metabolize nucleosides and nucleobases. Wild-type murine MVECs express mainly the ENT1 subtype with only trace levels of ENT2. These cells also have a Na(+)-independent equilibrative nucleobase transport mechanism for hypoxanthine (ENBT1). In the ENT1(-/-) cells, there is no change in ENT2 or ENBT1, resulting in a very low level of nucleoside uptake in these cells, but a high capacity for nucleobase accumulation. Whereas there were no significant changes in nucleoside transporter subtype expression, there was a dramatic increase in adenosine deaminase and adenosine A(2a) receptors (both transcript and protein) in the ENT1(-/-) tissues compared with WT. These changes in adenosine deaminase and A(2a) receptors likely reflect adaptive cellular mechanisms in response to reduced adenosine flux across the membranes of ENT1(-/-) cells. Our study also revealed that mouse MVECs have a nucleoside/nucleobase transport profile that is more similar to human MVECs than to rat MVECs. Thus mouse MVECs from transgenic animals may prove to be a useful preclinical model for studies of the effects of purine metabolite modifiers on vascular function.

Skeletal Muscle-Specific Activation of Gq Signaling Maintains Glucose Homeostasis.

Skeletal muscle (SKM) insulin resistance plays a central role in the pathogenesis of type 2 diabetes. Because G-protein-coupled receptors (GPCRs) represent excellent drug targets, we hypothesized that activation of specific functional classes of SKM GPCRs might lead to improved glucose homeostasis in type 2 diabetes. At present, little is known about the in vivo metabolic roles of the various distinct GPCR signaling pathways operative in SKM. In this study, we tested the hypothesis that selective activation of SKM Gq signaling can improve SKM glucose uptake and whole-body glucose homeostasis under physiological and pathophysiological conditions. Studies with transgenic mice expressing a Gq-linked designer GPCR selectively in SKM cells demonstrated that receptor-mediated activation of SKM Gq signaling greatly promoted glucose uptake into SKM and significantly improved glucose homeostasis in obese, glucose-intolerant mice. These beneficial metabolic effects required the activity of SKM AMPK. In contrast, obese mutant mice that lacked both Gαq and Gα11 selectively in SKM showed severe deficits in glucose homeostasis. Moreover, GPCR-mediated activation of Gq signaling also stimulated glucose uptake in primary human SKM cells. Taken together, these findings strongly suggest that agents capable of enhancing SKM Gq signaling may prove useful as novel antidiabetic drugs.

DREADD technology reveals major impact of Gq signalling on cardiac electrophysiology.

AIMS: Signalling via Gq-coupled receptors is of profound importance in many cardiac diseases such as hypertrophy and arrhythmia. Nevertheless, owing to their widespread expression and the inability to selectively stimulate such receptors in vivo, their relevance for cardiac function is not well understood. We here use DREADD technology to understand the role of Gq-coupled signalling in vivo in cardiac function. METHODS AND RESULTS: We generated a novel transgenic mouse line that expresses a Gq-coupled DREADD (Dq) in striated muscle under the control of the muscle creatine kinase promotor. In vivo injection of the DREADD agonist clozapine-N-oxide (CNO) resulted in a dose-dependent, rapid mortality of the animals. In vivo electrocardiogram data revealed severe cardiac arrhythmias including lack of P waves, atrioventricular block, and ventricular tachycardia. Following Dq activation, electrophysiological malfunction of the heart could be recapitulated in the isolated heart ex vivo. Individual ventricular and atrial myocytes displayed a positive inotropic response and arrhythmogenic events in the absence of altered action potentials. Ventricular tissue sections revealed a strong co-localization of Dq with the principal cardiac connexin CX43. Western blot analysis with phosphor-specific antibodies revealed strong phosphorylation of a PKC-dependent CX43 phosphorylation site following CNO application in vivo. CONCLUSION: Activation of Gq-coupled signalling has a major impact on impulse generation, impulse propagation, and coordinated impulse delivery in the heart. Thus, Gq-coupled signalling does not only modulate the myocytes' Ca2+ handling but also directly alters the heart's electrophysiological properties such as intercellular communication. This study greatly advances our understanding of the plethora of modulatory influences of Gq signalling on the heart in vivo.

Characterization of mENT1Delta11, a novel alternative splice variant of the mouse equilibrative nucleoside transporter 1.

Mammalian cells require specific transport mechanisms for the cellular uptake and release of endogenous nucleosides such as adenosine, and nucleoside analogs used in chemotherapy. We have identified a novel splice variant of the mouse equilibrative nucleoside transporter, mENT1, that results from the exclusion of exon 11 during pre-RNA processing. This variant encodes a truncated protein (mENT1Delta11) missing the last three transmembrane domains of the full-length mENT1. The mENT1Delta11 transcript and protein were found to be differentially distributed among tissues relative to full-length mENT1. PK15-NTD (nucleoside transport deficient) cells were transfected with mENT1 or mENT1Delta11 and assessed for nucleoside transport function. No significant differences were observed between the mENT1 and mENT1Delta11 in terms of transport function or inhibitor binding affinity. PK15-mENT1Delta11 transfected cells bound the ENT1 probe [3H]nitrobenzylthioinosine (NBMPR) with high affinity and mediated the cellular accumulation of both [3H]2-chloroadenosine and [3H]uridine. The only significant differences between the mENT1 variants were that mENT1Delta11 could not be photolabeled with [3H]NBMPR and that mENT1Delta11 was insensitive to the transporter-modifying effects of N-ethylmaleimide. These data suggest that the last three transmembrane domains of mENT1 are not necessary for transport activity, but this region does contain the cysteines responsible for the sensitivity of mENT1 to sulfhydryl reagents, and the residues important for covalent modification of the protein with NBMPR. These results provide important guidelines for future mutagenesis studies aimed at elucidating the tertiary structure of the ENT1 protein and the domains involved in inhibitor binding and substrate translocation.

Loss of equilibrative nucleoside transporter 1 in mice leads to progressive ectopic mineralization of spinal tissues resembling diffuse idiopathic skeletal hyperostosis in humans.

Diffuse idiopathic skeletal hyperostosis (DISH) is a noninflammatory spondyloarthropathy, characterized by ectopic calcification of spinal tissues. Symptoms include spine pain and stiffness, and in severe cases dysphagia and spinal cord compression. The etiology of DISH is unknown and there are no specific treatments. Recent studies have suggested a role for purine metabolism in the regulation of biomineralization. Equilibrative nucleoside transporter 1 (ENT1) transfers hydrophilic nucleosides, such as adenosine, across the plasma membrane. In mice lacking ENT1, we observed the development of calcified lesions resembling DISH. By 12 months of age, ENT1(-/-) mice exhibited signs of spine stiffness, hind limb dysfunction, and paralysis. Micro-computed tomography (µCT) revealed ectopic mineralization of paraspinal tissues in the cervical-thoracic region at 2 months of age, which extended to the lumbar and caudal regions with advancing age. Energy-dispersive X-ray microanalysis of lesions revealed a high content of calcium and phosphorus with a ratio similar to that of cortical bone. At 12 months of age, histological examination of ENT1(-/-) mice revealed large, irregular accumulations of eosinophilic material in paraspinal ligaments and entheses, intervertebral discs, and sternocostal articulations. There was no evidence of mineralization in appendicular joints or blood vessels, indicating specificity for the axial skeleton. Plasma adenosine levels were significantly greater in ENT1(-/-) mice than in wild-type, consistent with loss of ENT1--a primary adenosine uptake pathway. There was a significant reduction in the expression of Enpp1, Ank, and Alpl in intervertebral discs from ENT1(-/-) mice compared to wild-type mice. Elevated plasma levels of inorganic pyrophosphate in ENT1(-/-) mice indicated generalized disruption of pyrophosphate homeostasis. This is the first report of a role for ENT1 in regulating the calcification of soft tissues. Moreover, ENT1(-/-) mice may be a useful model for investigating pathogenesis and evaluating therapeutics for the prevention of mineralization in DISH and related disorders.

Hypoxanthine uptake and release by equilibrative nucleoside transporter 2 (ENT2) of rat microvascular endothelial cells.

The cardioprotective actions of adenosine are terminated by its uptake into endothelial cells with subsequent metabolism through hypoxanthine to uric acid. This process involves xanthine oxidase-mediated generation of reactive oxygen species (ROS), which have been implicated in the vascular dysfunction observed in ischemia-reperfusion injury. The equilibrative nucleoside transporter, ENT2, mediates the transfer of hypoxanthine into cells. We hypothesize that ENT2 also mediates the cellular release of hypoxanthine, which would limit the amount of intracellular hypoxanthine available for xanthine oxidase-mediated ROS production. Rat microvascular endothelial cells (MVECs) were isolated from skeletal muscle by lectin-affinity purification. The transport of [(3)H]hypoxanthine was assessed using an oil-stop method, and hypoxanthine metabolites were identified by thin-layer chromatography. MVECs accumulated hypoxanthine with a K(m) of 300 microM and a V(max) of 2.8 pmol microl(-1) s(-1). ATP-depleted cells loaded with [(3)H]hypoxanthine released the radiolabel with kinetics similar to that obtained for [(3)H]hypoxanthine influx. The uptake and release of [(3)H]hypoxanthine were both blocked by ENT2 inhibitors with similar order of potency. Thus, ENT2 mediates both the influx and efflux of hypoxanthine. Inhibition of ENT2 in MVECs might be expected to increase the amount of intracellular hypoxanthine available for metabolism by xanthine oxidase and enhance the intracellular production of ROS.

Nucleoside and nucleobase transporters of primary human cardiac microvascular endothelial cells: characterization of a novel nucleobase transporter.

Levels of cardiovascular active metabolites, like adenosine, are regulated by nucleoside transporters of endothelial cells. We characterized the nucleoside and nucleobase transport capabilities of primary human cardiac microvascular endothelial cells (hMVECs). hMVECs accumulated 2-[3H]chloroadenosine via the nitrobenzylmercaptopurine riboside-sensitive equilibrative nucleoside transporter 1 (ENT1) at a V(max) of 3.4 +/- 1 pmol.microl(-1).s(-1), with no contribution from the nitrobenzylmercaptopurine riboside-insensitive ENT2. Inhibition of 2-chloroadenosine uptake by ENT1 blockers produced monophasic inhibition curves, which are also compatible with minimal ENT2 expression. The nucleobase [3H]hypoxanthine was accumulated within hMVECs (K(m) = 96 +/- 37 microM; V(max) = 1.6 +/- 0.3 pmol.microl(-1).s(-1)) despite the lack of a known nucleobase transport system. This novel transporter was dipyridamole-insensitive but could be inhibited by adenine (K(i) = 19 +/- 7 microM) and other purine nucleobases, including chemotherapeutic analogs. A variety of other cell types also expressed the nucleobase transporter, including the nucleoside transporter-deficient PK(15) cell line (PK15NTD). Further characterization of [3H]hypoxanthine uptake in the PK15NTD cells showed no dependence on Na(+) or H(+). PK15NTD cells expressing human ENT2 accumulated 4.5-fold more [3H]hypoxanthine in the presence of the ENT2 inhibitor dipyridamole than did PK15NTD cells or hMVECs, suggesting trapping of ENT2-permeable metabolites. Understanding the nucleoside and nucleobase transporter profiles in the vasculature will allow for further study into their roles in pathophysiological conditions such as hypoxia or ischemia.

Differential regulation of mouse equilibrative nucleoside transporter 1 (mENT1) splice variants by protein kinase CK2.

Nucleosides are accumulated by cells via a family of equilibrative transport proteins (ENTs). An alternative splice variant of the most common subtype of mouse ENT (ENT1) has been identified which is missing a protein kinase CK2 (casein kinase 2) consensus site (Ser(254)) in the central intracellular loop of the protein. We hypothesized that this variant (mENT1a) would be less susceptible to modulation by CK2-mediated phosphorylation compared to the variant containing the serine at position 254 (mENT1b). Each splice variant was transfected into nucleoside transporter deficient PK15 cells, and stable transfectants assessed for their ability to bind the ENT1-selective probe [(3)H]nitrobenzylthioinosine (NBMPR) and to mediate the cellular uptake of [(3)H]2-chloroadenosine, with or without treatment with the CK2 selective inhibitor, 4,5,6,7-tetrabromobenzotriazole (TBB). mENT1a had a higher affinity for NBMPR relative to mENT1b - measured both directly by the binding of [(3)H]NBMPR, and indirectly via inhibition of [(3)H]2-chloroadenosine influx by NBMPR. Furthermore, incubation of mENT1b-expressing cells with 10 microM TBB for 48 h decreased both the K(D) and B(max) of [(3)H]NBMPR binding, as well as the V(max) of 2-chloroadenosine uptake, whereas similar treatment of mENT1a-expressing cells with TBB had no effect. PK15 cells transfected with hENT1, which has Ser(254), was similar to mENT1b in its response to TBB. In conclusion, inhibition of CK2 activity, or deletion of Ser(254) from mENT1, enhances transporter affinity for the inhibitor, NBMPR, and reduces the number of ENT1 proteins functioning at the level of the plasma membrane.