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. 2013 Aug 22;154(11):4136–4145. doi: 10.1210/en.2013-1357

GPER Deficiency in Male Mice Results in Insulin Resistance, Dyslipidemia, and a Proinflammatory State

Geetanjali Sharma 1, Chelin Hu 1, Jonathan L Brigman 1, Gang Zhu 1, Helen J Hathaway 1, Eric R Prossnitz 1,✉
PMCID: PMC3800768  PMID: 23970785

Abstract

Estrogen is an important regulator of metabolic syndrome, a collection of abnormalities including obesity, insulin resistance/glucose intolerance, hypertension, dyslipidemia, and inflammation, which together lead to increased risk of cardiovascular disease and diabetes. The role of the G protein-coupled estrogen receptor (GPER/GPR30), particularly in males, in these pathologies remains unclear. We therefore sought to determine whether loss of GPER contributes to aspects of metabolic syndrome in male mice. Although 6-month-old male and female GPER knockout (KO) mice displayed increased body weight compared with wild-type littermates, only female GPER KO mice exhibited glucose intolerance at this age. Weight gain in male GPER KO mice was associated with increases in both visceral and sc fat. GPER KO mice, however, exhibited no differences in food intake or locomotor activity. One-year-old male GPER KO mice displayed an abnormal lipid profile with higher cholesterol and triglyceride levels. Fasting blood glucose levels remained normal, whereas insulin levels were elevated. Although insulin resistance was evident in GPER KO male mice from 6 months onward, glucose intolerance was pronounced only at 18 months of age. Furthermore, by 2 years of age, a proinflammatory phenotype was evident, with increases in the proinflammatory and immunomodulatory cytokines IL-1β, IL-6, IL-12, TNFα, monocyte chemotactic protein-1, interferon γ-induced protein 10, and monokine induced by interferon gamma and a concomitant decrease in the adipose-specific cytokine adiponectin. In conclusion, our study demonstrates for the first time that in male mice, GPER regulates metabolic parameters associated with obesity and diabetes.

Over the past several decades, obesity and the associated increases in diabetes and cardiovascular disease have become a global health crisis (1). The pathologies associated with these diseases fall under the umbrella of metabolic syndrome, which includes insulin resistance/glucose intolerance, pancreatic dysfunction, hypertension, dyslipidemia, and inflammation. Obesity and a chronic proinflammatory state have also been linked to an increased incidence of multiple cancers (2, 3). As a result, there is an urgent need to identify novel molecular targets and therapeutic agents capable of preventing or limiting the development and consequences of these metabolic abnormalities.

Estrogen [17β-estradiol (E2)] is an important regulator of adiposity and insulin sensitivity in the body (4, 5). Premenopausal women exhibit higher insulin sensitivity compared with postmenopausal women, and the incidence of maladies associated with metabolic syndrome is lower in premenopausal women compared with men of similar age (6–8). Postmenopausal women exhibit an increased tendency toward insulin resistance and visceral fat deposition (9, 10).

Similarly, mouse models mimicking menopause (surgical ovariectomy) show an increased incidence of metabolic syndrome-associated abnormalities as in postmenopausal women, suggesting a critical role for estrogen in ameliorating these pathophysiological changes (6, 11). Consistent with these observations, estrogen replacement in postmenopausal women improves insulin sensitivity and leads to a reduction in body fat, patterns confirmed in ovariectomized mice supplemented with E2 (11, 12). Perhaps surprisingly, male mice, which exhibit an increased tendency to gain weight on a high-calorie diet compared with females, are also responsive to estrogen and estrogen mimetics (13, 14). For example, treatment of mice of both sexes with the selective estrogen receptor modulator acolbifene resulted in decreased fat accumulation and lower body weight (14). Similarly, using a novel therapeutic agent consisting of an E2-glucagon-like-peptide-1 conjugate, which aids in targeted delivery of E2, weight reduction and improved glucose tolerance were observed in male mice (15).

Finally, studies in mice have shown that neonatal or adult exposure to environmental estrogens such as xenoestrogens or phytoestrogens (eg, bisphenol A, diethylstilbestrol, and genistein), which are believed to act through estrogen receptors, can lead to adiposity with commensurate metabolic disturbances (16, 17). In addition to the quantity of fat, E2 also regulates the site of fat deposition (18), with increased abdominal fat in an E2-reduced environment (ie, in men and postmenopausal women) being linked to multiple health risks such as insulin resistance, diabetes, inflammation, and cardiovascular complications (1).

Adipose tissue, in addition to serving as a reservoir of fat, can alter the immunological, metabolic, and endocrine milieu, via secretion of various hormones (such as adiponectin) or adipokines (eg, proinflammatory cytokines such as TNFα, IL-1β) that in turn regulate adiposity through the modulation of glucose and fat metabolism (19, 20). Adiponectin levels are inversely correlated with body fat and insulin resistance, with obese individuals displaying severely reduced levels (21, 22), commensurate with decreased insulin sensitivity, fatty acid oxidation, energy expenditure, and hepatic glucose production (23). Furthermore, increased adipose mass leads to the secretion of proinflammatory cytokines that can activate stress pathways to bring about insulin resistance (24). The fact that ovariectomy and menopause result in insulin resistance with a concomitant increase in tissue inflammation further suggests that E2 regulates insulin sensitivity and inflammation (25, 26).

The effects of E2 have traditionally been attributed to the classical nuclear estrogen receptors (ERs), ERα and ERβ, which predominantly regulate transcription (27). Loss of E2 (as in postmenopausal women) or ER function (as in ERα knockout mice) results in decreased insulin sensitivity, increased abdominal fat accumulation, impaired glucose tolerance, and systemic inflammation (28, 29). However, recent evidence also suggests a role for the G protein-coupled estrogen receptor (GPER; previously named GPR30) in metabolic regulation (30). Mice lacking GPER are more likely to develop streptozotocin-induced diabetes compared with wild type (WT) mice (31). In addition, a role for GPER in islet survival is suggested by the protective effect of E2 in double-knockout (KO) mice lacking both ERα and ERβ treated with streptozotocin (31). Finally, the GPER-selective agonist G-1 (32) attenuates β-cell death in the MIN6 pancreatic β-cell line as well as in mouse and human islets (31) and promotes islet survival following transplantation in a murine model of type 1 diabetes (33). Mice deficient in GPER gain excess weight (34) and 6-month-old female GPER KO mice are glucose intolerant compared with WT mice (35). Interestingly, the latter study reported that male GPER KO mice exhibited normal glucose tolerance at that age. In the current study, we tested the hypothesis that with age, GPER deficiency in males will also recapitulate multiple aspects of metabolic syndrome, including obesity, dyslipidemia, insulin resistance, glucose intolerance, and inflammation.

Materials and Methods

Animals

C57BL/6 mice from Harlan Laboratories and GPER KO mice were maintained as previously described (36). GPER KO mice were provided by Jan Rosenbaum (Proctor & Gamble) and were described previously (37). Mice were backcrossed 10 generations onto C57BL/6 mice and housed at the animal research facility at the University of New Mexico Health Sciences Center. Animals were maintained under a controlled temperature of 22–23°C with a 12-hour light, 12-hour dark cycle and fed a normal chow ad libitum. All procedures were carried out in accordance with the National Institutes of Health Guide for the Humane Care and Use of Laboratory Animals and approved by the University of New Mexico Institutional Animal Care and Use Committee. Only male animals were used for this study; mice were euthanized between 10:00 am and 2:00 pm.

Food intake and home cage locomotor activity

For food intake studies, 4-month-old WT and GPER KO mice were placed in individual cages for 8 weeks and provided food and water ad libitum; food intake was measured weekly and expressed as grams of food consumed per day per mouse. Home cage locomotor activity was measured in WT and GPER KO mice to assess activity in a nonaversive environment. Briefly, mice were individually housed in a standard cage with food and water ad libitum and left undisturbed for a 24-hour acclimation period. Horizontal activity was then automatically measured and recorded for 48 hours by photocell beam breaks using the PAS-Homecage system (San Diego Instruments).

Fat measurements and magnetic resonance imaging (MRI)

Animals were weighed periodically, and epididymal and perirenal fat depots were carefully dissected and weighed after the animals were euthanized. Tibia length was measured after clearing of soft tissue. Body fat was also quantified by MRI on a Biospec 4.7T (Bruker Corp) with the following parameters: field of view, 4 cm × 4 cm; echo time, 13 milliseconds; repetition time, 5000 milliseconds; matrix, 256 × 256. Images were subjected to further analysis for separation of fat/water content by the two-point Dixon method (38). Six transverse sections per mouse from the same region of the middle abdomen were analyzed for total and sc fat content using ImageJ (National Institutes of Health).

Measurement of fasting plasma glucose, insulin, cholesterol, and triglycerides

Mice were fasted for 10 hours prior to the animals being euthanized, and blood was collected by cardiac puncture. Subsequently, glucose, insulin, triglycerides, and cholesterol [low density lipoprotein (LDL) and high density lipoprotein (HDL)] were measured in the plasma. Glucose was measured using the ReliOn Confirm glucose monitoring system (Relion Corp). Insulin was measured using a mouse insulin ELISA kit (Mercodia) as per the manufacturer's instructions. Cholesterol and triglyceride concentrations were determined at IDEXX RADIL diagnostic testing.

Measurement of adiponectin and cytokines

Nonfasted mice were euthanized and plasma samples were analyzed for adiponectin using a mouse adiponectin ELISA kit (R&D Systems). Plasma levels of a panel of cytokines were measured using the mouse cytokine magnetic 20-plex kit (Invitrogen) according to the manufacturer's protocol.

Glucose (GTT) and insulin tolerance tests (ITT)

For GTT and ITT determinations, mice were fasted for 4 hours. Glucose (2 g/kg body weight) or insulin (0.5 U/kg body weight) was then injected ip, and blood glucose levels were determined at regular time intervals from tail nicks.

Statistical analysis

Significance was determined by a Student's t test using GraphPad Prism version 5.00 for Windows (GraphPad Software). For GTT and ITT analyses, the area under the curve was calculated for individual mice, and genotypes were then compared using t tests. A value of P ≤ .05 was considered significant.

Results

GPER KO mice accumulate fat at multiple sites

Male GPER KO mice were consistently heavier than their WT counterparts at all ages examined in this study (by 7% at 6 mo; 18% at 12 mo; 13% at 18 mo; and 16% at 24 mo; Figure 1A). Body weights of each genotype increased until 18 months of age (with the GPER KO mice reaching a plateau by 12 mo), followed by a decline in both genotypes at advanced age (24 mo), consistent with previous observations in WT C57BL/6 mice (39). To rule out the possibility that increases in overall weight in the GPER KO mice were due to an increase in the size of mice, tibia length was measured and found to be similar for WT and GPER KO mice (WT: 18.58 ± 0.09 mm; KO: 18.62 ± 0.12 mm; P = .79). MRI studies revealed an increase in both total (43%) and sc fat (32%) in the GPER KO mice compared with WT mice (Figure 1, B and C). Measurement of individual fat pad weights confirmed that both perirenal (Figure 1D) and epididymal (Figure 1E) fat depots were substantially increased in GPER KO mice, by 34% and 40%, respectively.

Figure 1.

Figure 1.

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Increased adiposity in GPER KO mice. A, Body weights of WT (filled circles) and GPER KO (open circles) male mice at different ages. B, Representative body and MRI images from 1-year-old WT and GPER KO mice. C, Quantitation of total and sc fat content from individual transverse MRI image slices of 1-year-old WT and GPER KO mice (mean ± SEM, n = 3). Using six transverse sections from the same region of the middle abdomen, average fat per slice was determined for total and sc fat content using ImageJ. Perirenal (D) and epididymal (E) fat depots were weighed after the dissection of 1-year-old WT and GPER KO mice. GPER KO mice were heavier, with an increase in fat deposition at multiple sites in the body.

To determine whether differences in food intake or activity could account for the differences in body weight and fat mass, we determined food intake and home cage locomotor activity in a cohort of 4- to 6-month-old mice, an age before there is a substantial divergence in body weight between the GPER KO and WT mice. Food intake measurements revealed no difference between GPER KO and WT mice, suggesting that adiposity in GPER KO mice did not develop as a result of increased caloric intake (Figure 2A). Additionally, home cage locomotor experiments with 4- to 6-month-old mice showed that there was no significant difference between genotypes with respect to total activity level over a 48-hour period. To determine whether GPER KO mice might exhibit a decrease in activity as they age, we also assessed home cage locomotor activity in 1-year-old mice; as with younger mice, there were no differences between WT and GPER KO mice (data not shown). This indicates that WT and GPER KO mice do not differ in the level or pattern of locomotor activity in a familiar environment, leading to the conclusion that increased body weight and adipose also do not result from reduced activity in the GPER KO mice (Figure 2B).

Figure 2.

Figure 2.

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GPER KO mice do not differ in food intake or locomotor activity. A, Food intake was measured weekly in 4-month-old WT and GPER KO mice for 8 weeks. B, Home cage locomotor activity was measured in 4-month-old WT and GPER KO mice over a 48-hour period by photocell beam break analysis. Values are expressed as mean ± SEM (n = 5); no significant differences in either food intake or locomotor activity were detected.

GPER KO mice exhibit altered lipid metabolism

Analysis of plasma from fasted animals revealed an altered lipid profile in the GPER KO mice. In 1-year-old animals, the levels of circulating total cholesterol and triglycerides were significantly higher in the GPER KO mice when compared with the WT mice (Figure 3). Total cholesterol in the GPER KO mice was 51% higher than in the WT mice, whereas triglycerides levels were 71% higher in the GPER KO mice. Although GPER KO mice exhibited an approximately 200% increase in LDL levels, which bordered on significance (P = .08), HDL levels were clearly not different between the two genotypes.

Figure 3.

Figure 3.

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GPER KO mice display an altered lipid profile. Plasma lipid profiles (total cholesterol, triglyceride, LDL, and HDL levels) were determined in 1-year-old WT and GPER KO mice. Mice were fasted overnight prior to blood collection, euthanized, and subsequently lipids were measured in plasma. Values are expressed as mean ± SEM (n = 5–7). GPER KO mice had higher circulating cholesterol and triglyceride levels compared with their WT counterparts.

Insulin resistance and glucose intolerance in mice lacking GPER

Although glucose tolerance at 6 months of age was similar between the WT and GPER KO mice (Figure 4A), as the animals aged (12 and 18 mo of age), a decline in glucose tolerance was observed in the GPER KO mice, reaching significance at 18 months (Figure 4A). However, insulin tolerance tests revealed that the GPER KO mice were insulin resistant at all ages tested (Figure 4B), including at 6 months when glucose tolerance was normal. Furthermore, basal glucose levels (0 time point) in the GPER KO mice were similar to those of WT animals at all ages, suggesting a compensatory mechanism in GPER KO mice counteracts peripheral insulin resistance. To explore this further, we evaluated fasting levels of glucose and insulin in 1-year-old mice. Although GPER KO mice exhibited normal fasting blood glucose levels, fasting insulin levels were elevated (Figure 5), likely accounting for the normal basal fasting glucose levels. The extent of insulin resistance was quantified by the homeostatic model assessment (HOMA-IR) (40) and found to be higher in the GPER KO mice (Figure 5). Conversely, using the quantitative insulin sensitivity check index (QUICKI) (41), we demonstrated that GPER KO mice exhibited decreased insulin sensitivity compared with WT mice (Figure 5).

Figure 4.

Figure 4.

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GPER KO mice exhibit glucose intolerance and insulin resistance. GTTs (A) and ITTs (B) were performed on WT (filled circles, n = 8) and GPER KO (open circles, n = 10) mice at 6, 12, and 18 months of age. After a 4-hour fast, glucose (2 g/kg body weight for GTT) or insulin (0.5 U/kg body weight for ITT) was injected ip after which blood glucose levels were monitored over a 2-hour period. The area under the curve was calculated for each individual mouse. Results are expressed as mean ± SEM. Although GPER KO mice displayed insulin resistance by 6 months of age, glucose tolerance remained near normal up to 12 months of age, after which glucose tolerance was impaired.

Figure 5.

Figure 5.

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Insulin sensitivity is diminished in GPER KO mice. Glucose and insulin levels from 1-year-old WT (n = 5) and GPER KO (n = 7) mice were determined in plasma after an overnight fast. Fasting glucose and insulin levels were further used to assess insulin sensitivity by HOMA-IR and the QUICKI. Values are expressed as mean ± SEM. GPER KO mice exhibited lower insulin sensitivity compared with their WT counterparts because they required higher fasting insulin levels to maintain normal blood glucose levels.

GPER KO mice exhibit increased proinflammatory cytokines and decreased adiponectin

Because peripheral inflammation may emerge as a secondary consequence of weight gain, we determined the cytokine profiles of 1- and 2-year-old mice. Plasma cytokine profiles of 1-year-old mice did not reveal any significant differences between WT and GPER KO mice (data not shown). However, by 2 years of age, proinflammatory cytokines, IL-1β, IL-6, and TNFα, were significantly higher in the GPER KO mice, with monocyte chemotactic protein-1 (MCP-1) showing a trend toward increased levels (Figure 6). Three additional immunomodulatory cytokines, IL-12, interferon-γ-induced protein 10 (IP-10), and monokine, induced by γ-interferon (MIG) were also higher in the GPER KO mice. Conversely, adiponectin was lower in the GPER KO mice, consistent with obesity and a proinflammatory phenotype (Figure 6). No differences were detected in the levels of six other cytokines (macrophage inflammatory protein 1α, chemokine (CXC motif) ligand-1/keratinocyte-derived chemoattractant (CXCL1/KC), granulocyte-macrophage colony stimulating factor, IL-2, IL-5, and IL-13); vascular endothelial growth factor and IL-17 were lower in GPER KO mice (data not shown). Interferon-γ, basic fibroblast growth factor, IL-1α, IL-4, and IL-10 were undetectable in all samples.

Figure 6.

Figure 6.

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Proinflammatory cytokine levels are elevated in GPER KO mice. Proinflammatory cytokines, IL-1β, TNFα, IL-6, and MCP-1, and other immunomodulatory cytokines, IL-12, IP-10, and MIG, as well as the adipose-specific cytokine adiponectin were measured in the plasma from WT (n = 6) and GPER KO (n = 7) mice at 2 years of age. Values are expressed as mean ± SEM. GPER KO mice revealed a proinflammatory phenotype (as evidenced by increased IL-1β, TNFα, IL-6, IL-12, IP-10, and MIG-1 levels with a concomitant decrease in adiponectin levels).

Discussion

Steroid hormones, and E2 in particular, are important physiological modulators of the complex events that regulate metabolism and body weight (42). E2 actively regulates energy expenditure, adiposity, and insulin sensitivity as well as the survival and function of pancreatic β-cells (4, 25, 31). Ovariectomized WT mice and ERα KO mice exhibit an increase in body weight, insulin resistance, and inflammation (29, 43). Parallel observations in humans reveal that postmenopausal women are more prone to visceral weight gain and metabolic dysfunction (7, 8). In both humans and mice, the effects of hormone replacement therapy clearly indicate a protective role for E2. Until recently, such effects were thought to be mediated solely by the classical estrogen receptors, ERα and ERβ, via genomic pathways (27). Although a role for ERα in the modulation of adiposity has been clearly demonstrated, increasing evidence now reveals a contribution of nongenomic/rapid signaling events in mediating the effects of E2 in physiology and metabolism that appear to be mediated in part by GPER (30, 36). In the present study, we characterized the effect of GPER deficiency on adiposity, insulin sensitivity, and inflammation in male mice.

GPER KO mice in our study were consistently heavier than their WT counterparts. Importantly, we observed the greatest weight difference at an age beyond that examined in other studies, suggesting an age-dependent effect on the relative weight gain in male GPER KO mice. A recent report of increased overall size in GPER KO mice (44) disagrees with our measurements that detected no difference in tibia length, which is also consistent with other studies (35). Although not all studies of GPER KO mice have reported weight increases, differences in the extent of weight gain, age of onset, and sex dependence could be the result of confounding sampling, environmental, or genetic factors (45). Based on our results, we conclude that overall weight gain in GPER KO mice is due to increased fat content within multiple sites including epidydimal, perirenal, and sc depots. Furthermore, the lack of differences in food intake or locomotion activity between WT and GPER KO mice indicates that weight gain in GPER KO mice is not a consequence of increased food consumption or decreased activity. In this respect, there is a limited parallel between GPER KO and ERα KO mice; although the latter do not display increased food intake compared with WT mice, they do display lower activity and energy expenditure (43). In postmenopausal women, weight gain results in part from a decrease in metabolic energy expenditure (46). Thus, GPER KO mice, although not less active, may exhibit a decrease in metabolic energy expenditure that contributes to increased weight gain.

Weight gain results from and leads to an altered metabolic phenotype, manifested in GPER KO mice as elevated levels of fasting cholesterol, triglycerides, and insulin, although fasting glucose levels remained normal. Interestingly, our results are similar to those from male ERα KO mice, which are also obese and display an abnormal lipid profile (47). In our study, loss of GPER yielded higher plasma levels of cholesterol and triglycerides, with the LDL increase bordering on significance, altogether suggesting that GPER has a function in the regulation of lipid metabolism.

Hyperinsulinemia is an additional factor that may lead to dyslipidemia (48). Our results showed that the GPER KO mice required higher insulin levels to maintain normal glucose levels compared with their WT counterparts. This hyperinsulinemia in GPER KO mice may also contribute to the elevated levels of circulating lipids. An increase in adiposity, specifically abdominal adiposity, is strongly correlated with insulin resistance and glucose intolerance (49). The lack of glucose intolerance in young GPER KO mice likely results from increased, compensatory insulin secretion, mimicking early stages of insulin resistance in humans, in whom pancreatic β-cells produce more insulin, resulting in hyperinsulinemia to maintain normal glucose levels (50). Whether this occurs through hypertrophy or hyperplasia of pancreatic-β cells/islets would be of interest to determine in future studies. Fasting glucose and insulin levels were used to calculate both HOMA and QUICKI values, which confirmed that GPER KO mice were insulin resistant. Furthermore, GTT and ITT revealed that, although GPER KO mice were insulin resistant from an early age, they were glucose tolerant at younger ages and developed impaired glucose tolerance only at an advanced age. The observation of progressive glucose intolerance, despite early tolerance, could result from the cumulative effects of dyslipidemia and worsening insulin resistance with age as well as ensuing systemic inflammation, similar to the progression in humans where obesity leads to insulin resistance, dyslipidemia, chronic inflammation, and eventually diabetes.

With age, GPER KO mice exhibited increased levels of proinflammatory cytokines (TNFα, IL-1β, MCP-1, and IL-6) with a concomitant decrease in adiponectin, changes that were not yet evident in 1-year-old mice. Levels of additional immunomodulatory cytokines such as IP-10, IL-12, and MIG were also higher in the GPER KO mice. Although IL-6 can act as either a proinflammatory or an antiinflammatory cytokine (by decreasing TNFα levels) (51), because TNFα levels were also elevated in our study, IL-6 is likely acting in a proinflammatory capacity in GPER KO mice. Because abdominal fat produces more IL-6 than sc fat (52), increased visceral fat in GPER KO mice may represent a source of this elevated IL-6. Postmenopausal women and ERα KO mice also exhibit increased serum levels of proinflammatory cytokines (43, 53), most likely derived from excess adipose tissue. Plasma adiponectin levels in obese adults are lower than in normal subjects (54), and in mice, adiponectin administration reverses insulin resistance and weight gain (55, 56). However, insulin also regulates adiponectin, although in a complex manner, with acute insulin stimulation increasing adiponectin gene expression but prolonged insulin exposure leading to a decrease in the adiponection expression (20, 21). It is therefore likely that the hyperinsulinemia that develops in the GPER KO mice eventually leads to the down-regulation of adiponectin in these mice.

It is important to note that the effects of E2 and therefore also multiple E2 receptors on metabolism are complex and likely multifactorial and interconnected, with both direct and indirect effects that may positively and negatively impact overall metabolic status. A comprehensive analysis of these complex interactions is beyond the scope of any single study but clearly merits further investigation. Interestingly, similarities in the metabolic phenotypes observed upon deletion of either ERα or GPER in mice (increased adipose/weight gain, dyslipidemia, insulin resistance/glucose intolerance, and inflammation) suggest a requirement for both receptors to maintain normal metabolism. A role for GPER is also suggested by the observations that mice lacking both ERα and ERβ maintain antidiabetic effects upon E2 stimulation (31). The similar phenotypic metabolic alterations of ERα KO and GPER KO mice further suggest that many aspects of metabolic regulation by E2 may involve the activities of both ERα and GPER, with loss of either resulting in metabolic dysregulation. Whereas the end metabolic effects of deficiency in either receptor may be similar, the mechanisms and pathways involved may be very different (57). Although both receptors may exhibit synergism to regulate metabolism in WT mice, the expression of either ERα or GPER alone may yield a partial compensatory activity in the absence of the other receptor. Although the effects of GPER on peripheral tissues important in glucose and fat metabolism (ie, adipose, muscle, and liver) have not been examined, the effects of GPER deficiency suggest that GPER may exert direct antiadipogenic effects on these tissues. Finally, although phenotypic similarities exist between ERα KO and GPER KO mice in terms of the metabolic parameters investigated here, it should be noted that these mice exhibit significant differences in reproductive capacity with ERα KO mice being infertile, whereas GPER KO maintain normal reproductive function. These differences are corroborated by the GPER-selective ligand G-1, which exerts little estrogenic effect on the uterus in terms of imbibition and epithelial proliferation (58).

In conclusion, our data demonstrate for the first time that GPER-deficient male mice exhibit the following: 1) an overall increase in fat content throughout the body, 2) altered lipid profiles, 3) insulin resistance, and 4) a proinflammatory phenotype. Because recent reviews highlight ERs as emerging targets for the treatment of obesity and diabetes (6), GPER-selective agonists may contribute to the therapeutic arsenal for treating obesity, diabetes, and related metabolic and inflammatory diseases without the deleterious/feminizing effects of E2 on reproductive and other organs.

Acknowledgments

We thank Dr Natalie Adolphi (University of New Mexico) for assistance with the MRI image analysis and Dr Kevin Caldwell for his helpful discussions. G.S., C.H., and J.L.B. carried out the experiments; G.Z. analyzed the MRI images; G.S. and E.R.P. designed the experiments, analyzed the data, and wrote the manuscript; and C.H. and H.J.H. reviewed and edited the manuscript; and all of the authors had the final approval of the submitted version of the manuscript.

This work was supported by National Institutes of Health Grants R01CA127731 and R01CA163890 (to E.R.P.) and by dedicated health research funds from the University of New Mexico School of Medicine (to E.R.P.). The MRI images were generated at the University of New Mexico Brain Imaging Center supported by National Institutes of Health Centers of Biomedical Research Excellence Grant P30 GM10340. Statistical support was provided by the University of New Mexico Clinical and Translational Science Center supported by National Institutes of Health Grant 1ULRR31977.

Disclosure Summary: E.R.P. is an inventor on US patent number 7,875,721. The other authors have nothing to disclose.

Footnotes

Abbreviations:
E2
17β-estradiol
ER
estrogen receptor
GPER
G protein-coupled estrogen receptor
GTT
glucose tolerance test
HDL
high-density lipoprotein
HOMA-IR
homeostatic model assessment for insulin resistance
IP-10
interferon-γ-induced protein 10
ITT
insulin tolerance test
KO
knockout
LDL
low-density lipoprotein
MCP-1
monocyte chemotactic protein-1
MIG
monokine induced by γ-interferon
MRI
magnetic resonance imaging
QUICKI
quantitative insulin sensitivity check index
WT
wild type.

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