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Units of Drug Target Discovery (U.E.A.M., J.D.-N., C.Ow., B.O., L.M.F.L.-L.), Vascular Physiology (M.F.G., K.S., P.H., B.-O.N.), and Circulation Physiology (P.-O.G.), Department of Experimental Medical Science, Lund University, SE-22184 Lund, Sweden; Division of Endocrinology and Diabetes (S.A.S., M.F.G., A.W., I.L.), Department of Clinical Sciences, Malmö, Lund University, SE-20502 Malmö, Sweden; Center for Bone Research at the Sahlgrenska Academy (S.W., N.A., C.Oh.), Department of Internal Medicine, Göteborg University, SE-41345 Göteborg, Sweden; The Jackson Laboratory (C.J.R.), Bar Harbor, Maine 04401; Department of Biochemistry (M.L.A.), The University of Texas Health Science Center, San Antonio, Texas 78229; and Oxford Centre for Diabetes Endocrinology and Metabolism (P.R.), University of Oxford, Churchill Hospital, Oxford OX3 7LJ, United Kingdom
Address all correspondence and requests for reprints to: L. M. Fredrik Leeb-Lundberg, Department of Experimental Medical Science, Lund University, BMC, A12, SE-22184 Lund, Sweden. E-mail: fredrik.leeb-lundberg{at}med.lu.se.
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Introduction |
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The best-documented mechanism for estrogen action involves the binding to two types of nuclear estrogen receptors (ERs) named ER
and ERβ, which are ligand-activated transcription factors that stimulate the expression of several target genes (10, 11). Whereas many important metabolic estrogenic events occur through this mechanism, several estrogen responses are membrane dependent and frequently occur via G protein-coupled pathways, which cannot be explained by the aforementioned nuclear mechanism (12). Membrane-associated versions of ER complexed with signaling and scaffolding molecules capable of acting via G proteins have been identified that may explain in part such events (10, 11).
G protein-coupled receptor (GPR) 30, was recently shown to bind E2 in vitro with high affinity and, therefore, proposed to be the cognate ligand for this receptor (13, 14). Originally cloned from a Burkitt’s lymphoma cell line (15), GPR30 was found to mediate E2-stimulated increases in cAMP and intracellular Ca2+, and mediate E2-promoted proliferative signaling in vitro in an estrogen-sensitive but ER-negative breast cancer cell line (13, 14, 16, 17) and human endometrial cells (18) via transactivation of the epidermal growth factor receptor. GPR30 is also required for progestin inhibition of MCF-7 breast cancer cell proliferation (19), and for transcriptional glucocorticoid activity (20). Despite significant progress on the function of GPR30 in vitro (21), the physiological role of GPR30 in vivo is unknown, and it remains to be determined whether GPR30 is an ER also in vivo.
We developed a mouse model in which the GPR30 gene locus was completely disrupted that allowed us to investigate the metabolic role(s) of GPR30 in greater detail. Our results show that GPR30 is necessary for normal insulin production, glucose homeostasis, skeletal growth, and blood pressure in females, which is consistent with the role of estrogen in these events. Importantly, GPR30 is required for E2-stimulated insulin release in both female and male mice but is critical for normal glucose homeostasis only in females.
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Materials and Methods |
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). A total of 45 µg targeting vector was linearized with Not 1 and electroporated into 3 x 106 R1 embryonic stem (ES) cells using a Bio-Rad gene pulser (Bio-Rad Laboratories, Inc., Hercules, CA). The cells were subjected to G418 selection (350 µg/ml; GIBCO/BRL, Invitrogen Corp., Carlsbad, CA), and the resulting colonies were analyzed for homologous recombination and intactness using PCR and Southern blot analysis (Fig. 1). Of 192 clones surviving G418 selection, 22 had undergone homologous recombination in the GPR30 locus, and 12 clones also had an intact upstream loxP site. The GPR30 gene was deleted by transient expression of cAMP response element-recombinase. The chosen targeting strategy resulted in deletion of the whole GPR30 open-reading frame. Three clones were injected into C57BL/6 blastocysts, which were implanted into pseudopregnant females. To obtain heterozygous F1 offspring, the chimeric male founders were crossed with C57BL/6 females. Only one founder produced fertile germ line offspring. The deletion was backcrossed six generations into the C57BL/6 genetic background. Genotyping of the mice was done using PCR on tail biopsies and was subsequently confirmed by Southern blot hybridization (Fig. 1). The disruption of the gene was finally confirmed at the mRNA level by quantitative real-time PCR (qRTPCR) analysis of GPR30 expression (Fig. 2C) (data not shown). All primers used in targeting GPR30 are listed in the supplemental data, which are published as supplemental data on The Endocrine Society’s Journals Online web site at http://endo. endojournals.org.
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RIA
RIA kits were used to assay insulin (AID Diagnostika GmbH, Straßberg, Germany), glucagon (Euro-Diagnostica AB, Malmö, Sweden), and E2 (DiaSorin Inc., Stillwater, MN). An ELISA kit was used to assay leptin (Chrystal Chem Inc., Downers Grove IL). Serum IGF-I was measured by a double-antibody RIA using a two-step method as described (23). There is no cross-reactivity with IGF-II, and the interassay coefficient of variation for C57Bl/6J female mouse serum is 5%.
Pancreatic islet preparation and insulin and glucagon secretion
Mice were killed by cervical dislocation. Pancreatic islets were then isolated after retrograde injection of a collagenase solution (Sigma-Aldrich Corp., St. Louis, MO) via the bile-pancreatic duct essentially as described previously (24). After digestion, individual islets were isolated at room temperature using a stereomicroscope. Islets for RNA isolation were directly frozen at –70 C, whereas islets for hormone release studies and electrophysiology were placed in Krebs-Ringer bicarbonate buffer (pH 7.4) supplemented with 10 mM HEPES, 0.1% fatty acid-free BSA (Roche Molecular Biochemicals, Indianapolis, IN), and 1 mM glucose. For electrophysiology, the β-cells were isolated by triculation in Ca2+-free buffer as described previously (25).
For insulin and glucagon secretion, the aforementioned buffer was replaced with fresh buffer containing either 1 or 20 mM glucose, and the islets were further incubated in the absence or presence of tolbutamide (400 µM), K+ (50 mM), or E2 (5 µM) for 60 min at 37 C unless otherwise stated. Each vial contained 12 islets in 1 ml buffer, gassed with 95% O2/5% CO2 to obtain constant pH and oxygenation, and incubated in a shaking incubator (30 cycles per minute). An aliquot of the medium was removed immediately after incubation and frozen at –20 C for subsequent assay of insulin and glucagon.
RNA isolation and reverse transcription for qRTPCR
Total RNA from tissues was isolated using the guanidinium isothiocyanate method, and from islets and mesenteric arteries using the Absolutely RNA Microprep kit (Stratagene, La Jolla, CA). cDNA synthesis was performed by reserve transcription for 50 min at 50 C in a 20-µl reaction mix containing 500 ng total RNA (islets and mesenteric arteries) or 5 µg total RNA (tissues), 10 mM dithiothreitol, 5 mM MgCl2, 0.5 µg oligo(deoxythymidine)12–18, 0.5 mM deoxynucleotide triphosphate (dNTP), 40 U RNaseOUT (Invitrogen), and 50 U Superscript II RT (Invitrogen).
qRTPCR
qRTPCR was performed in a LightCycler system (Roche Molecular Biochemicals) using the SYBR Green I detection method. The reactions were performed in a total volume of 20 µl containing 5 µl diluted cDNA (1:20), ddH2O (negative control), or purified gene-specific PCR product (for standard curves), 1x PCR buffer (Invitrogen), 3–3.5 mM MgCl2, 0.5 µM of each primer (supplemental data), 200 µM of each dNTP, 0.5 µg/µl BSA, 1:30,000 dilution of SYBR Green I, and 0.5 U platinum Taq DNA polymerase (Invitrogen). QuantiTect primers were used for glucose transporter (GLUT) 2 and glucokinase (QIAGEN, Inc., Valencia, CA). After denaturation at 94 C for 2.5 min, a total of 40 cycles were run (each 10 sec at 96 C, 10 sec at 57 C, 20 sec at 72 C). Melting curve profiles were analyzed and the specificity of the bands further verified by electrophoresis on agarose gels. To generate external standards, specific PCR products for each gene were purified using QIAquick gel extraction kit (QIAGEN). The copy number was calculated based on the measured concentration at 260 nm, and serial 10-fold dilutions were made in ultrapure water.
For detection of ER, ERβ, and GPR30 in islets and ER and GPR30 in mesenteric arteries, a pre-PCR was performed before the qRTPCR. The reaction was performed in a total volume of 50 µl containing 10 µl (1:4) diluted cDNA, 0.3 µM forward and reverse primer of ER, ERβ, and β2-microglobulin (supplemental data), 0.2 mM dNTP, 3.5 mM MgCl2, and 0.05 U platinum Taq DNA polymerase. After denaturation at 95 C for 5 min, a total of 15 cycles was run (each 30 sec at 95 C, 30 sec at 58 C, 30 sec at 72 C). A total of 5 µl diluted (1:4) pre-PCR product was used in the qRTPCR. β2-Microglobulin was used for normalization of data in all experiments.
Glucose tolerance test
Glucose (5.6 mmol/kg body weight) was dissolved in 0.9% NaCl and delivered by ip injection. Blood sampling and determination of plasma levels of insulin, glucagon, and glucose were performed as described previously (24, 26).
Islet electrophysiology
β-Cell exocytosis was measured as an increase in membrane capacitance using the standard whole cell configuration of the patch clamp technique as described previously (25). The cell culture medium was supplemented with 5 mM glucose, and the intracellular solution contained 0.1 mM cAMP to enhance the exocytotic response.
Blood pressure and heart rate recordings
Mice were anesthetized with sodium pentobarbital (80 mg/kg) given ip. A catheter was inserted into the jugular vein to allow iv administration of the nitric oxide (NO) synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME) (Sigma-Aldrich) and the prostacyclin synthase inhibitor tranylcypromine (ICN Biomedicals, Inc., Costa Mesa, CA). Mean arterial blood pressure (MAP) and heart rate were monitored with a Grass transducer coupled to a heparinized catheter inserted into the femoral artery. The body temperature was maintained at 37 C throughout the experiment via a feedback-controlled heating pad.
Morphometric analysis
Thoracic aorta and second-generation mesenteric resistance arteries were fixed, embedded, cryosectioned, and stained with hematoxylin-eosin. For each aorta and mesenteric artery, five to six and 11–29 sections were examined, respectively. Lumen was calculated by measuring the intima luminal border (Image-Pro Plus Software; Media Cybernetics, Inc., Bethesda, MD) assuming a circular shape of the lumen in vivo. Media were defined as the distance between internal and external elastic lamina.
Statistics
Summarized data are presented as means ± SEM. The Student’s two-tailed t test for unpaired data was used to evaluate statistical significance. P values less than 0.05 were regarded as statistically significant. Comparison of islet insulin release between female and male GPR30(–/–) and GPR30(+/+) mice was done by two-way ANOVA.
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), pancreatic islets, and mesenteric resistance arteries (Fig. 2C). On the other hand, GPR30 expression was below the detection level in liver and various fat deposits, including brown, gonadal, retroperitoneal, and inguinal fat (Fig. 2A). GPR30 mRNA expression in the male mouse was similar to that in the female in several tissues (Fig. 2B). To investigate the physiological role(s) of GPR30 and the function of this receptor as a target for E2, a mouse model was developed in which the GPR30 gene was completely deleted (Fig. 1, see Materials and Methods). Figure 2C shows that GPR30(–/–) mice lacked expression of GPR30 mRNA as exemplified with brain, including the pituitary (data not shown), skeletal muscle, pancreatic islets, and mesenteric arteries. On the other hand, ER and ERβ were expressed at normal levels in GPR30(–/–) mice (Fig. 2C).
Female GPR30(–/–) mice have impaired metabolic phenotypes
Female GPR30(–/–) mice exhibited an age-dependent reduction in body weight (–9.6% at 19 wk; P < 0.05), whereas the weight gain of male GPR30(–/–) mice was not significantly different from male GPR30(+/+) mice (Fig. 2D). The reduced female body weight was associated with a proportional reduction in skeletal growth because both the axial skeleton (crown-rump length; Fig. 2E) and the appendicular skeleton (femur length; Fig. 2F) were significantly reduced. In contrast, male GPR30(–/–) mice displayed normal skeletal development (Fig. 2, E and F). A proportional growth disturbance in female GPR30(–/–) mice was further supported by the unaffected organ weight to body weight ratio for several major organs investigated, including uterus, lung, kidney, spleen, liver, heart, and skeletal muscle (Table 1). The reduced female body weight was not due to a reduced amount of body fat because both the white adipose tissue deposit weight (Table 1), weight of individual deposits (data not shown), and serum leptin level were unchanged in both females and males (Table 2).
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). This difference was not observed when comparing male GPR30(–/–) and GPR30(+/+) mice. More detailed analysis revealed that female GPR30(–/–) mice were glucose intolerant with both a more rapid early increase in and a sustained elevation of the plasma glucose level (Fig. 3A), which remained significantly elevated when compensating for the hyperglycemia (3 min, P < 0.001; 45 min, P < 0.05). On the other hand, male GPR30(–/–) mice had normal glucose tolerance (Fig. 3B). Further analysis showed that the impaired glucose tolerance of the female GPR30(–/–) mice was associated with a significantly diminished first-phase insulin response to the glucose challenge (Fig. 3C) and depressed glucagon levels at the 10-min time point (Fig. 3D). Thus, the absence of GPR30 yields an impaired glucose tolerance specifically in females, which is associated with an attenuated first-phase insulin response.
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), which was confirmed using the tail-cuff method (data not shown). On the other hand, at 9 months the MAP was elevated (23%) in female GPR30(–/–) mice (Fig. 2H). No difference was observed in the heart rate between these mice at either 6 or 9 months of age (Fig. 2I).
Because estrogens have been shown to participate in each of the physiological events perturbed in female GPR30(–/–) mice, affected serum E2 levels may be the reason for the phenotypical changes in these mice. However, no difference in the E2 level was observed between female GPR30(–/–) and GPR30(+/+) mice (Table 2). The GH/IGF-I axis is a major determinant of body weight and tissue growth (27). Analysis of the serum IGF-I level revealed a tendency toward a reduced level at 7 wk of age and a significantly reduced level at 6 months of age in female GPR30(–/–) mice compared with female GPR30(+/+) mice (Table 2). In contrast, male GPR30(–/–) mice had a normal IGF-I level. Thus, reduced serum IGF-I levels may contribute to some of the metabolic disturbances in the female GPR30(–/–) mice.
The impaired glucose tolerance in female GPR30(–/–) mice in vivo correlates with decreased insulin expression and insulin release in vitro in isolated pancreatic islets
To address in more detail the role of GPR30 in the impaired insulin response and glucose tolerance in vivo, we next investigated insulin expression and release in vitro in pancreatic islets isolated from female and male GPR30(+/+) and GPR30(–/–) mice. Insulin release from isolated islets was stimulated 10.2-fold by 20 mM glucose in both female and male GPR30(+/+) mice (compare Fig. 4, A and B). Tolbutamide (400 µM) and high K+ (50 mM) were also stimulatory under low-glucose (1 mM) and high-glucose (20 mM) conditions in both gender. Islets from female GPR30(–/–) mice had significantly reduced insulin release at both low (–35%) and high-glucose levels (–57%) (Fig. 4, A and B) even though the fold stimulation by glucose remained about the same (5.8-fold). Similar reductions in insulin release were also observed in response to tolbutamide and high K+ (Fig. 4, A and B), but again, their fold stimulation was similar in the GPR30(+/+) and GPR30(–/–) mice under low glucose (3.5-, 3.8-, 3.2-, and 2.8-fold, respectively) and high glucose (1.8-, 1.7-, 2.5-, and 2.5-fold, respectively). Female GPR30(–/–) mice had reduced islet insulin levels as determined both by the decrease in islet insulin I (–68%) (Fig. 4E) and insulin II (–45%) mRNA levels (Fig. 4F) and the decrease in insulin content (–34%) (Fig. 4G). On the other hand, β-cell exocytosis was normal in the female GPR30(–/–) mice, as measured by the depolarization-evoked increase in cell capacitance (
Cm) (Fig. 4H). Furthermore, the expression of two early mediators of glucose-stimulated insulin release in islets, GLUT2 and glucokinase, were not different between GPR30(+/+) and GPR30(–/–) mice (Fig. 4I). In addition, male GPR30(–/–) mice, which exhibited normal glucose tolerance and significantly less perturbation in insulin release than female GPR30(–/–) mice at 1 mM (P < 0.05) and 20 mM glucose (P < 0.001) (Fig. 4, A and B), had normal insulin levels (Fig. 4, E–G). No significant difference in islet insulin release at 1 mM glucose was observed in female GPR30(–/–) mice when the release was normalized to the islet insulin content, whereas a significant difference was still observed at 20 mM glucose (P < 0.001).
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, C and D). Glucagon release was slightly reduced in female GPR30(–/–) mice at both low (–18%) and high-glucose (–12%) levels but completely unaffected in male GPR30(–/–) mice (Fig. 4C). Unlike insulin, islet glucagon mRNA levels were normal in female GPR30(–/–) mice (Fig. 4J).
The estrogenic response on insulin release in vivo is absent in female GPR30(–/–) mice
Estrogens regulate insulin release, and both ovariectomy and menopause lead to perturbations in the release of insulin (28). To address if GPR30 is required for E2 regulation of insulin release in vivo, ovariectomized GPR30(+/+) and GPR30(–/–) mice were treated with and without a physiological concentration of E2 (70 ng/mouse · d) for 4 wk, a standard regimen to evaluate effects of E2. The serum insulin level and total pancreatic insulin content were then used as assay parameters for insulin release. E2 stimulated a significant increase (90%; P < 0.05) in the serum insulin level in ovariectomized GPR30(+/+) mice (Fig. 5A). This E2 response was completely absent in ovariectomized GPR30(–/–) mice. The pancreatic insulin content in ovariectomized GPR30(+/+) mice was not significantly different with and without E2 treatment (Fig. 5B). On the other hand, E2 treatment of ovariectomized GPR30(–/–) mice yielded a highly significant increase (50%; P < 0.001) in the total pancreatic insulin content. Because ER has previously been shown to protect islet integrity (29), and is expressed at unaltered levels in GPR30(–/–) mice (Fig. 2C), we attribute the observed increase in the pancreatic insulin content specifically in these mice to an ER-mediated mechanism, which in the absence of a concurrent GPR30-mediated E2 stimulation of insulin release results in an increase in the islet insulin content.
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), we next wished to determine whether the GPR30-dependent E2 response on insulin release observed in vivo is the result of E2 acting directly through GPR30 in pancreatic islets. Indeed, E2 acts on β-cells and -cells in vitro to stimulate insulin release and inhibit glucagon release, respectively, through membrane mechanisms that are thought not to involve ER (30, 31). To this end, islets from GPR30(+/+) and GPR30(–/–) mice were stimulated with E2. Application of E2 to islets from female GPR30(+/+) mice led to increases in insulin release and decreases in glucagon release under both low (88 and –41%, respectively) (Fig. 6, A and C) and high-glucose conditions (54 and –22%, respectively) (Fig. 6, B and D). Islets from male GPR30(+/+) also responded to E2 with similar increases in insulin release and decreases in glucagon release under low (51 and –37%, respectively) (Fig. 6, A and C) and high-glucose conditions (49 and –21%, respectively) (Fig. 6, B and D). On the other hand, islets from both female and male GPR30(–/–) mice, although responding to glucose, tolbutamide, and high K+, were completely devoid of insulin and glucagon responses to E2 (Fig. 6). The losses of these E2 responses in GPR30(–/–) mice were not due to a change in islet ER and ERβ mRNA expression, which were the same in GPR30(+/+) and GPR30(–/–) mice (Fig. 2C). Thus, islets express GPR30 through which E2 stimulates insulin release and inhibits glucagon release, respectively. Collectively, these results provide strong evidence that GPR30 is a physiologically relevant receptor for estrogen in vivo, and coupled to stimulation of insulin release in both females and males. However, this receptor seems to be critical for normal glucose homeostasis only in females.
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) and relative heart weight (Table 1) were unchanged in the female GPR30(–/–) mice, the increase in MAP in these mice (Fig. 2H) is most likely due to an increase in total vascular resistance rather than an increase in cardiac output. Intravenous infusion of L-NAME (10 mg/kg) to inhibit NO synthase led to similar increases in MAP in the GPR30(+/+) (23.6 ± 2.6 mm Hg, mean ± SEM, n = 4) and GPR30(–/–) mice (18.9 ± 2.2 mm Hg, mean ± SEM, n = 4). Infusion of tranylcypromine (0.1 mg/kg) to inhibit prostacyclin synthase subsequent to L-NAME infusion had no further effect (data not shown). Furthermore, acetylcholine relaxed aortic rings from GPR30(+/+) and GPR30(–/–) mice precontracted with high K+ equally well (data not shown). Thus, the elevated MAP in female GPR30(–/–) mice is apparently not due to vascular mechanisms related to the release of NO or prostacyclin.
GPR30 and ER are expressed in second-order mesenteric resistance arteries (Fig. 2C). Figure 7A shows representative images of arteries from GPR30(+/+) and GPR30(–/–) mice at age 9 months, an age at which MAP was increased (Fig. 2H). Assessing the dimensions of the arteries at this age, we found that the media thickness of the arteries was not different between the mice (Fig. 7B). On the other hand, the lumen circumference of the arteries was reduced (–23%) (Fig. 7C), and the media to lumen ratio was increased (45%) in the GPR30(–/–) mice (Fig. 7D). This structural alteration was specific to resistance arteries because no difference was observed in the dimensions of the thoracic aorta of GPR30(+/+) and GPR30(–/–) mice at this age (data not shown). No difference was observed in the arterial dimensions at 6 months of age (Fig. 7, B–D), an age at which MAP was not increased (Fig. 2H). Thus, the increased MAP in the female GPR30(–/–) mice is associated with a remodeling specifically of the mesenteric resistance arteries in these mice.
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The weight of the uterus was unaffected in GPR30(–/–) mice, indicating that GPR30 is not a crucial receptor for the estrogenic growth response of this tissue. This is in contrast to ER(–/–) mice, which have severely affected uteri (32), and ERβ(–/–) mice, which were recently shown to have a disturbed growth and differentiation of the uterine epithelium (33). On the other hand, female GPR30(–/–) mice also exhibited a proportional reduction in skeletal growth in line with the role of estrogens in this response (34, 35, 36). The reduced skeletal growth in female ER(–/–) mice correlated with reduced serum IGF-I levels (37), whereas the increased skeletal growth of female ERβ(–/–) mice was associated with increased serum IGF-I levels (38). Thus, IGF-I may be a mediator or indicator of estrogenic effects on skeletal growth during sexual maturation as initiated both via the two nuclear ERs, ER and ERβ, and via GPR30. Endocrine IGF-I is mainly synthesized in the liver (39, 40). Because GPR30 expression could not be detected in the liver, a direct regulation of IGF-I synthesis by this receptor in the liver is unlikely. GH is the main regulator of liver-produced IGF-I, and it remains to be determined whether GPR30 regulates GH secretion at either the hypothalamic or pituitary level.
Female GPR30(–/–) mice exhibited impaired glucose tolerance, which was associated with reduced glucose-stimulated insulin release both in vivo and in vitro. Islets from female GPR30(–/–) mice expressed normal levels of GLUT2 and glucokinase, two early mediators of glucose-stimulated insulin release. On the other hand, an apparent decrease in insulin expression was observed. In contrast, male GPR30(–/–) mice, which had only slightly impaired insulin release in vitro and apparently normal levels of insulin expression, exhibited normal glucose tolerance. Although decreased insulin levels is one possible reason for the reduced glucose tolerance in the female GPR30(–/–) mice, additional islet impairments in the insulin release mechanism are also possible. The glucose load was also associated with significantly decreased glucagon levels at the 10-min time point in these mice, which may be due to the elevated plasma glucose levels inhibiting glucagon release in these mice.
E2 is known to reverse the hyperglycemia, impaired glucose tolerance, reduced insulin, and increased glucagon release caused by ovariectomy and menopause (28, 41, 42, 43, 44, 45). Treatment of ovariectomized GPR30(+/+) mice with E2 at 70 ng/mouse · d for 4 wk led to an increase in the serum insulin level confirming that this hormone promotes insulin release at physiological concentrations in vivo. Deletion of GPR30 completely eliminated the increase in serum insulin in response to E2 in vivo, indicating that GPR30 is involved in this E2 response. Because GPR30 is expressed in pancreatic islets, as shown here, and that GPR30 has previously been proposed to be a high-affinity receptor for E2 (13, 14), we hypothesized that E2 may act directly through this receptor in islets to stimulate insulin release. E2 stimulated insulin release in vitro directly in islets isolated from GPR30(+/+) mice. On the other hand, E2 was completely unable to elicit this response in islets isolated from GPR30(–/–) mice, which are devoid of GPR30 expression but expresses normal levels of the nuclear ERs, ER and ERβ. Thus, we provide both in vivo and in vitro evidence that E2 stimulation of insulin release is dependent on GPR30 expressed in the islet. Our results are consistent with earlier observations that E2 acts directly on β-cells to trigger insulin release (30, 31). The fact that this E2 response is membrane dependent and insensitive to both of the antiestrogen ER ligands tamoxifen and ICI 182,780 (30, 31) suggests that it is not dependent on ER but rather on another receptor, possibly GPR30. Interestingly, the GPR30-dependent E2 response in vitro occurred at both low and high-glucose levels, making it different from some other cAMP-elevating agonists, which stimulate this response only at high-glucose levels. This may be explained by the fact that GPR30 elicits not only an elevation of cAMP but also an elevation of intracellular Ca2+, the latter that is directly linked to insulin release.
E2 also promoted an increase in total pancreatic insulin content in vivo in ovariectomized female GPR30(–/–) mice. This response is in apparent conflict with the observed decrease in basal islet insulin expression in the same but nonovariectomized mice. We suggest that the E2-promoted increase in the insulin content is mediated by ER and that this response becomes visible only in the absence of GPR30-dependent E2 stimulation of insulin release. ER is expressed at an unaltered level in islets from GPR30(–/–) mice. Furthermore, ER(–/–) mice do not develop any impaired insulin release (46), indicating that ER is not directly involved in this response. On the other hand, E2 maintains islet integrity by an antiapoptotic hypertrophic effect on the islets, which is mediated by ER (29). Therefore, it is reasonable to propose that the absence of GPR30-mediated E2 stimulation of insulin release could lead to a visible increase in the pancreatic insulin content. Whether the decrease in basal insulin levels in the female GPR30(–/–) mice is developmentally related or the result of deficient E2 signaling through islet GPR30 is unclear. This regulation may also involve GPR30-dependent variations in IGF-I production, as previously shown (47). In any case, E2-regulated insulin homeostasis in vivo is likely to be complex and the net result of multiple mechanisms. Thus, in vivo in ovariectomized GPR30(–/–) mice at 70 ng/mouse · d of E2 for 4 wk, ER may be the most visible mediator of the E2 effect on the pancreatic insulin content.
E2 also inhibited glucagon release from pancreatic islets in a GPR30-dependent manner. This action could be due to a direct effect of E2 through GPR30 expressed in -cells. Although E2 acts directly on -cells to inhibit glucagon release in a membrane-dependent and antiestrogen-insensitive manner (30, 31), this conclusion has to await the direct identification of GPR30 expression in these cells. Alternatively, E2 inhibition of glucagon release could be the indirect result of the elevated GABA within the islet, which has been shown to be coreleased with insulin from β-cells and inhibit glucagon release from -cells (48).
Female GPR30(–/–) mice exhibited an increased MAP at 9 months of age. This increase was not associated with a change in cardiac output but rather with an increase in the media to lumen ratio. Although such arterial remodeling is consistent with an increased peripheral vascular resistance, we cannot exclude the involvement of other factors such as changes in the water balance or the renin-angiotensin system. Estrogens elicit multiple and diverse effects on the cardiovascular system with both direct antiatherogenic effects on the blood vessel wall and systemic effects on coagulation and on vasoactive proteins (49). Neither NO release nor prostacyclin production contributes to the increased MAP in the GPR30(–/–) mice. GPR30 is expressed, together with ER, in mesenteric resistance arteries. However, whether or not the arterial remodeling in GPR30(–/–) mice is the result of deficient E2 signaling through GPR30 either directly in the arteries or via a deficient systemic factor, such as IGF-I, or a developmental defect requires further studies. Interestingly, mice with liver-specific IGF-I inactivation have increased blood pressure (50).
In summary, we demonstrate that GPR30 is required for normal bone growth, glucose homeostasis, and blood pressure in female mice. Importantly, GPR30 is essential for E2 stimulation of insulin release. Thus, GPR30 may be an interesting therapeutic target to exploit for the development of novel treatments of some estrogen-related metabolic diseases in females.
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mediates increase in islet insulin content in male mice in vitro and in vivo, which is consistent with our interpretation of the E2-promoted increase in insulin content in vivo observed specifically in GPR30–/– mice.
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Disclosure Statement: The authors have nothing to disclose.
First Published Online October 9, 2008
1 B.O. and L.M.F.L.-L. contributed equally to this study. 
Abbreviations: dNTP, Deoxynucleotide triphosphate; E2, 17β-estradiol; ER, estrogen receptor; ES, embryonic stem; GLUT, glucose transporter; GPR, G protein-coupled receptor; L-NAME, NG-nitro-L-arginine methyl ester; MAP, mean arterial blood pressure; NO, nitric oxide; qRTPCR, quantitative real-time PCR; RT, reverse transcriptase.
Received April 30, 2008.
Accepted for publication September 29, 2008.
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plays an important role in the regulation of glucose homeostasis in mice: insulin sensitivity in the liver. Diabetologia 49:588–597[CrossRef][Medline]-Cells of the endocrine pancreas: 35 years of research but the enigma remains. Endocr Rev 28:84–116. PloS ONE 3:e2069This article has been cited by other articles:
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  S. Liu and F. Mauvais-Jarvis Minireview: Estrogenic Protection of {beta}-Cell Failure in Metabolic Diseases Endocrinology, March 1, 2010; 151(3): 859 - 864. [Abstract] [Full Text] [PDF]
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 B. R. S. Broughton, A. A. Miller, and C. G. Sobey Endothelium-dependent relaxation by G protein-coupled receptor 30 agonists in rat carotid arteries Am J Physiol Heart Circ Physiol, March 1, 2010; 298(3): H1055 - H1061. [Abstract] [Full Text] [PDF]
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 M. Maggiolini and D. Picard The unfolding stories of GPR30, a new membrane-bound estrogen receptor J. Endocrinol., February 1, 2010; 204(2): 105 - 114. [Abstract] [Full Text] [PDF]
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 E. A. Ariazi, E. Brailoiu, S. Yerrum, H. A. Shupp, M. J. Slifker, H. E. Cunliffe, M. A. Black, A. L. Donato, J. B. Arterburn, T. I. Oprea, et al. The G Protein-Coupled Receptor GPR30 Inhibits Proliferation of Estrogen Receptor-Positive Breast Cancer Cells Cancer Res., February 1, 2010; 70(3): 1184 - 1194. [Abstract] [Full Text] [PDF]
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 S. Soriano, A. B. Ropero, P. Alonso-Magdalena, C. Ripoll, I. Quesada, B. Gassner, M. Kuhn, J.-A. Gustafsson, and A. Nadal Rapid Regulation of KATP Channel Activity by 17{beta}-Estradiol in Pancreatic {beta}-Cells Involves the Estrogen Receptor {beta} and the Atrial Natriuretic Peptide Receptor Mol. Endocrinol., December 1, 2009; 23(12): 1973 - 1982. [Abstract] [Full Text] [PDF]
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 A. Nadal, P. Alonso-Magdalena, S. Soriano, A. B. Ropero, and I. Quesada The role of oestrogens in the adaptation of islets to insulin resistance J. Physiol., November 1, 2009; 587(21): 5031 - 5037. [Abstract] [Full Text] [PDF]
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 S. Liu, C. Le May, W. P.S. Wong, R. D. Ward, D. J. Clegg, M. Marcelli, K. S. Korach, and F. Mauvais-Jarvis Importance of Extranuclear Estrogen Receptor-{alpha} and Membrane G Protein-Coupled Estrogen Receptor in Pancreatic Islet Survival Diabetes, October 1, 2009; 58(10): 2292 - 2302. [Abstract] [Full Text] [PDF]
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G. G J Hazell, S. T Yao, J. A Roper, E. R Prossnitz, A.-M. O'Carroll, and S. J Lolait Localisation of GPR30, a novel G protein-coupled oestrogen receptor, suggests multiple functions in rodent brain and peripheral tissues J. Endocrinol., August 1, 2009; 202(2): 223 - 236. [Abstract] [Full Text] [PDF]
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S. H. Lindsey, J. A. Cohen, K. B. Brosnihan, P. E. Gallagher, and M. C. Chappell Chronic Treatment with the G Protein-Coupled Receptor 30 Agonist G-1 Decreases Blood Pressure in Ovariectomized mRen2.Lewis Rats Endocrinology, August 1, 2009; 150(8): 3753 - 3758. [Abstract] [Full Text] [PDF]
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 C. Ohlsson and L. Vandenput The role of estrogens for male bone health Eur. J. Endocrinol., June 1, 2009; 160(6): 883 - 889. [Abstract] [Full Text] [PDF]
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E. R. Levin G Protein-Coupled Receptor 30: Estrogen Receptor or Collaborator? Endocrinology, April 1, 2009; 150(4): 1563 - 1565. [Full Text] [PDF]
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 G.-F. Qiao, B.-Y. Li, Y.-J. Lu, Y.-L. Fu, and J. H. Schild 17{beta}-Estradiol restores excitability of a sexually dimorphic subset of myelinated vagal afferents in ovariectomized rats Am J Physiol Cell Physiol, January 1, 2009; 297(3): C654 - C664. [Abstract] [Full Text] [PDF]
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) and GPR30(–/–) mice (n = 9) (
) after ip glucose injection (5.6 mmol/kg body weight). Plasma levels of glucose (A and B), insulin (C), and glucagon (D) were then measured. Values are means ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001 for GPR30(–/–) vs. GPR30(+/+) mice. ns, Not significant.
