Deletion of the G Protein-Coupled Receptor 30 Impairs Glucose Tolerance, Reduces Bone Growth, Increases Blood Pressure, and Eliminates Estradiol-Stimulated Insulin Release in Female Mice

In vitro studies suggest that the G protein-coupled receptor (GPR) 30 is a functional estrogen receptor. However, the physiological role of GPR30 in vivo is unknown, and it remains to be determined whether GPR30 is an estrogen receptor also in vivo. To this end, we studied the effects of disrupting the GPR30 gene in female and male mice. Female GPR30^(−/−) mice had hyperglycemia and impaired glucose tolerance, reduced body growth, increased blood pressure, and reduced serum IGF-I levels. The reduced growth correlated with a proportional decrease in skeletal development. The elevated blood pressure was associated with an increased vascular resistance manifested as an increased media to lumen ratio of the resistance arteries. The hyperglycemia and impaired glucose tolerance in vivo were associated with decreased insulin expression and release in vivo and in vitro in isolated pancreatic islets. GPR30 is expressed in islets, and GPR30 deletion abolished estradiol-stimulated insulin release both in vivo in ovariectomized adult mice and in vitro in isolated islets. Our findings show that GPR30 is important for several metabolic functions in female mice, including estradiol-stimulated insulin release.

The G protein-coupled receptor GPR30 maintains normal bone growth, glucose homeostasis, and blood pressure, and mediates estradiol-stimulated insulin release in female mice.

Estrogens, the most potent being 17β-estradiol (E2), participate in the normal development and maintenance of the female reproductive tissues, and continue to regulate several different physiological processes, including glucose, bone, and cardiovascular homeostasis, during the reproductive years. The decrease in the estrogen level at menopause increases the risk for the metabolic syndrome, hypertension, and osteoporosis (1–4), the former two being major risk factors for diabetes and cardiovascular disease, respectively. Estrogen replacement therapy reverses many of these complications (5–7), but the effects are complex and age dependent with potentially deleterious side effects. Indeed, estrogen-dependent cancers of both the breast and endometrium account for about 40% of all cancers in women (8). Thus, estrogens are central to female health and disease, making it essential to establish their molecular targets and actions (9).

The best-documented mechanism for estrogen action involves the binding to two types of nuclear estrogen receptors (ERs) named ERα 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 ERα 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.

Materials and Methods

Generation of GPR30^(−/−) mice

The targeting vector containing a lox-flanked mGPR30 ORF and a lox-flanked TkNeo cassette was assembled from a 129/SvJ BAC-clone (Genome Systems, Inc., St. Louis, MO). To provide negative selection, the long arm was flanked with a PGKDt-a expression cassette (Fig. 1). A total of 45 μg targeting vector was linearized with Not 1 and electroporated into 3 × 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.

Fig. 1

Generation of GPR30^(−/−) mice. A and B, Schematic representation of the GPR30 targeting vector and the vector recombination with the target gene. C, Positions of the probes and the different PCR primers that were used to characterize the targeted GPR30 gene. D, Southern blot analysis of BamHI-digested genomic DNA from targeted ES cell clone (lane 1), targeted ES cell clone after deletion (lane 2), and wild-type clone (lane 3). Arrows indicate 6.9-kb band from the targeted allele (a), 6.1-kb band from the untargeted allele (b), and 3.2-kb band from the targeted allele after disruption of the GPR30 gene (c). The DNA was hybridized with a 0.8-kb HindIII/EcoRI fragment from outside of the recombination section of the GPR30 gene (see c). E, Southern blot analysis of XbaI digested genomic DNA from GPR30^(+/+) and GPR30^(−/−) mice. The DNA was hybridized with a 1.7-kb XbaI fragment containing the GPR30 gene.

Fig. 2

GPR30 expression and metabolic phenotypes in GPR30^(+/+) and GPR30^(−/−) mice. Expression of GPR30 mRNA in various tissues from female (A) and male (B) GPR30^(+/+) mice (n = 3–4). C, Expression of GPR30, ERα, and ERβ mRNA in various tissues from female and male GPR30^(+/+) and GPR30^(−/−) mice (n = 3–4 brain and skeletal muscle preparations; n = 8–12 islet preparations; three pooled arterial preparations). D, Body weights of female and male GPR30^(+/+) and GPR30^(−/−) mice up to 19 wk of age (female GPR30^(+/+), n = 10; female GPR30^(−/−), n = 11; male GPR30^(+/+), n = 9; male GPR30^(−/−), n = 9). E, Crown-rump length of female and male GPR30^(+/+) and GPR30^(−/−) mice at 19 wk of age (female GPR30^(+/+), n = 26; female GPR30^(−/−), n = 25; male GPR30^(+/+), n = 21; male GPR30^(−/−), n = 23). F, Femur length of female and male GPR30^(+/+) and GPR30^(−/−) mice at 23 wk of age (female GPR30^(+/+), n = 7; female GPR30^(−/−), n = 8; male GPR30^(+/+), n = 6; male GPR30^(−/−), n = 9). G, Plasma glucose levels in female and male GPR30^(+/+) and GPR30^(−/−) mice at 6 months of age (female GPR30^(+/+), n = 7, female GPR30^(−/−), n = 8; male GPR30^(+/+), n = 8, male GPR30^(−/−), n = 9). H, MAP in female GPR30^(+/+) and GPR30^(−/−) mice at 6 and 9 months of age (n = 4–6 per group). I, Heart rates in female GPR30^(+/+) and GPR30^(−/−) mice at 6 and 9 months of age (n = 4–6 per group). Values are means ± sem. *, P < 0.05; **, P < 0.01; ***, P < 0.001 for GPR30^(−/−)vs. GPR30^(+/+) of the same gender. GPR30^(+/+) mice (filled bars and symbols), GPR30^(−/−) mice (open bars and symbols). nd, Not detected; ns, not significant.

Animals and animal treatment

The mice were housed in a standard animal facility under controlled temperature (22 C) and photoperiod (12 h light, 12 h dark), and fed standard phytoestrogen-free pellet diet ad libitum. Littermate or age-matched C57BL/6 mice were used as a control group. For the experiments investigating the estrogenic responses in ovariectomized mice, 3-month-old female GPR30^(−/−) and GPR30^(+/+) mice were ovariectomized and then treated either with placebo or with 70 ng/mouse · d E2 for 4 wk using slow-release pellets (Innovative Research of America, Sarasota, FL). The E2 dose was chosen according to a study demonstrating affected estrogenic responses in a mouse model with inactivation of steroid receptor coactivator-1 (22). Animal care was in accordance with institutional guidelines. All animal experiments had been approved by the local Ethical Committees for Animal Research.

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), 1× 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 ERα, ERβ, and GPR30 in islets and ERα 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 ERα, 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.

Results

Tissue expression of GPR30

The expression of GPR30 mRNA in the female mouse was high in brain, skeletal muscle, and kidney, and lower in lung, heart (Fig. 2A), 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, ERα 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).

Table 1

Relative organ weights in female GPR30^(+/+) and GPR30^(−/−) mice

Organ	GPR30(+/+) (n = 7)	GPR30(−/−) (n = 8)
Uterus/BW (%)	0.28 ± 0.05a	0.38 ± 0.04
WAT/BW (%)	5.39 ± 0.96	4.25 ± 0.53
Lung/BW (%)	0.60 ± 0.03	0.58 ± 0.04
Kidney/BW (%)	1.12 ± 0.03	1.10 ± 0.04
Spleen/BW (%)	0.41 ± 0.05	0.44 ± 0.03
Liver/BW (%)	4.21 ± 0.07	3.99 ± 0.17
Heart/BW (%)	0.48 ± 0.03	0.48 ± 0.02
Muscle (quadriceps)/BW (%)	0.71 ± 0.01	0.77 ± 0.02

Values are expressed as percent organ weight/body weight (BW). Numbers of animals are indicated in parentheses. WAT, Dissected white adipose tissue, including gonadal, retroperitoneal, and inguinal fat deposits.

^a

Wet weights of different organs normalized to body weight in 6-month-old female GPR30^(+/+) and GPR30^(−/−) mice and given as means ± sem.

Table 2

Hormone levels in female and male GPR30^(+/+) and GPR30^(−/−) mice

Hormone	Age	Females		Males
		GPR30(+/+) (n = 6–7)	GPR30(−/−) (n = 7–8)	GPR30(+/+) (n = 6–8)	GPR30(−/−) (n = 9)
Estradiol (pg/ml)	7 wk	na	na	na	na
	6 months	15.5 ± 1.2a	14.6 ± 1.0	nd	nd
Testosterone (ng/ml)	7 wk	na	na	na	na
	6 months	nd	nd	2.82 ± 1.51	1.23 ± 0.67
Leptin (ng/ml)	7 wk	2.39 ± 0.49	1.71 ± 0.15	1.81 ± 0.48	1.70 ± 0.20
	6 months	8.96 ± 2.35	8.14 ± 1.58	8.69 ± 5.10	10.50 ± 4.63
IGF-I (ng/ml)	7 wk	306 ± 26	269 ± 12	242 ± 10	248 ± 22
	6 months	220 ± 8	195 ± 5b	209 ± 7	208 ± 9
Insulin (μU/ml)	6 months	12.5 ± 3.1	16.3 ± 2.1	na	na
Glucagon (ng/liter)	6 months	178 ± 10.7	170 ± 8	na	na
Glucose (mmol/liter)	6 months	8.4 ± 0.4	9.5 ± 0.2c	9.3 ± 0.4	8.3 ± 0.4

Numbers of animals are indicated in parentheses. na, Not assayed; nd, not detected.

^a

Values were obtained from serum or plasma of GPR30^(+/+) and GPR30^(−/−) mice and given as means ± sem.

^b

P < 0.05 for GPR30^(−/−)vs. GPR30^(+/+) mice.

^c

P < 0.01 for GPR30^(−/−)vs. GPR30^(+/+) mice.

At age 6 months, female GPR30^(−/−) mice fed ad libitum had slightly but significantly elevated plasma glucose levels (13%; P < 0.01) compared with female GPR30^(+/+) mice (Fig. 2G). 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.

Fig. 3

Glucose homeostasis in vivo in GPR30^(+/+) and GPR30^(−/−) mice. Glucose tolerance tests were performed on 6-month-old female (A, C, and D) and male (B) GPR30^(+/+) (n = 8) (•) 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.

At 6 months, no difference was observed in the MAP between female GPR30^(+/+) and GPR30^(−/−) mice as determined invasively in anesthetized mice (Fig. 2H), 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 (ΔC_m) (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).

Fig. 4

Hormone expression and release in vitro in pancreatic islets from GPR30^(+/+) and GPR30^(−/−) mice. Pancreatic islets isolated from 6-month-old female and male GPR30^(+/+) and GPR30^(−/−) mice were stimulated with and without 400 μm tolbutamide (T) or 50 mm K⁺ (K) at 1 (A and C) and 20 mm (B and D) glucose (G), and then assayed for insulin (A and B) and glucagon (C and D) as indicated. Data points are from 10 measurements, each including 12 islets. E and F, Insulin I (n = 8–12) and insulin II (n = 8–10) mRNA. G, Insulin content (n = 4). H, Increases in cell capacitance (ΔC_m) in β-cells evoked by 500-msec depolarizations from −70 to 0 mV (n = 12–20). I, GLUT2 and glucokinase mRNA (n = 4–5). J, Glucagon mRNA (n = 8–10). Values are means ± sem. *, P < 0.05; **, P < 0.01; ***, P < 0.001. GPR30^(+/+) (filled bars) and GPR30^(−/−) mice (open bars). ns, Not significant.

As expected, islet glucagon release was reduced by high glucose and by tolbutamide under basal conditions in both female (−40 and −34%, respectively) and male (−39 and −40%, respectively) GPR30^(+/+) mice (Fig. 4, 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 ERα 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 ERα-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.

Fig. 5

Estrogenic regulation of insulin release in vivo in GPR30^(+/+) and GPR30^(−/−) mice. Three-month-old female GPR30^(+/+) and GPR30^(−/−) mice were ovariectomized and then treated with placebo (ovx) or 70 ng/mouse · d E2 (ovx + E2) for 4 wk. Serum insulin (ovx GPR30^(+/+), n = 23; ovx GPR30^(−/−), n = 16; ovx + E2 GPR30^(+/+), n = 22; ovx + E2 GPR30^(−/−), n = 16) (A) and total pancreatic insulin (ovx GPR30^(+/+), n = 12; ovx GPR30^(−/−), n = 8; ovx + E2 GPR30^(+/+), n = 12; ovx + E2 GPR30^(−/−), n = 8) (B) was then assayed. Values are means ± sem. *, P < 0.05; ***, P < 0.001. GPR30^(+/+) (filled bars) and GPR30^(−/−) mice (open bars). ns, Not significant.

The estrogenic responses on insulin and glucagon release in vitro are absent in female and male GPR30^(−/−) mice

Because islets express GPR30 (Fig. 2C), 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 α-cells in vitro to stimulate insulin release and inhibit glucagon release, respectively, through membrane mechanisms that are thought not to involve ERα (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 ERα 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.

Fig. 6

Estrogenic regulation of insulin and glucagon release in vitro in pancreatic islets from GPR30^(+/+) and GPR30^(−/−) mice. Pancreatic islets isolated from 6-month-old female and male GPR30^(+/+) and GPR30^(−/−) mice were stimulated with and without 5 μm E2 (E2) at 1 (A and C) and 20 mm (B and D) glucose (G), and then assayed for the release of insulin (A and B) and glucagon (C and D) as indicated. Data points are from 10 measurements, each including 12 islets. Values are means ± sem. *, P < 0.05; **, P < 0.01; ***, P < 0.001. GPR30^(+/+) (filled bars) and GPR30^(−/−) mice (open bars). ns, Not significant.

The increased blood pressure in female GPR30^(−/−) mice is associated with altered resistance arterial structure

Considering that the heart rate (Fig. 2I) 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 ERα 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.

Fig. 7

Structure of mesenteric resistance arteries in female GPR30^(+/+) and GPR30^(−/−) mice. A, Representative images of second-order mesenteric resistance arteries from female GPR30^(+/+) and GPR30^(−/−) mice as indicated. Morphometric measurements of media thickness (B), lumen circumference (C), and media to lumen ratio (D) of arteries from animals at 6 (n = 5) and 9 months of age (n = 3). Sections (11–29) of at least five arteries from each animal were measured. Data are expressed per animal (= n), and values are means ± sem. *, P < 0.05 for GPR30^(+/+)vs. GPR30^−/− mice. GPR30^(+/+) (filled bars) and GPR30^−/− mice (open bars). In A, images were converted to gray scale. ns, Not significant.

Discussion

Here, we show that deletion of the gene for the putative ER GPR30 results in hyperglycemia and impaired glucose tolerance, reduced skeletal growth, and increased blood pressure in female mice. The impaired glucose tolerance in vivo is associated with decreased first-phase insulin release, which may be due in part to reduced islet insulin expression and, consequently, decreased total glucose-stimulated insulin release. Importantly, E2-stimulated insulin release is abolished in GPR30^(−/−) mice. Because serum E2 levels are apparently normal in female GPR30^(−/−) mice, we propose that the lack of E2 signaling either directly or indirectly through GPR30 contributes to the impaired insulin response and glucose metabolism in these mice.

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 ERα^(−/−) 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 (34–36). The reduced skeletal growth in female ERα^(−/−) 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, ERα 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, 41–45). 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, ERα 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 ERα 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 ERα and that this response becomes visible only in the absence of GPR30-dependent E2 stimulation of insulin release. ERα is expressed at an unaltered level in islets from GPR30^(−/−) mice. Furthermore, ERα^(−/−) mice do not develop any impaired insulin release (46), indicating that ERα 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 ERα (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, ERα 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 α-cells. Although E2 acts directly on α-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 α-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 ERα, 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.

Note Added in Proof

While this paper was in review, a study was published (51) showing that ERα 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.

Acknowledgments

This work was supported by funds from European Foundation for the Study of Diabetes/Servier, GS Development in Malmö, The Swedish Diabetes Association, Konsul Thure Carlssons Minne Foundation, Magnus Bergvalls Foundation, Diabetes Association in Malmö, Syskonen Svenssons Foundation, Anders Otto Swärds Foundation, Royal Physiographic Society in Lund, Lars Hiertas Minne Foundation, Torsten och Ragnar Söderbergs Foundations, The Swedish Research Council, and the Faculty of Medicine at Lund University in part through the Vascular Wall prioritized program. P.R. is a Wolfson-Royal Merit Award Research Fellow. U.E.A.M., A.W., and S.W. were supported by postdoctoral fellowships from the Swedish Society for Medical Research.

Disclosure Statement: The authors have nothing to disclose.

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

Author notes

^*

B.O. and L.M.F.L.-L. contributed equally to this study.