ORIGINAL ARTICLE G Protein–Coupled Receptor Kinase 2 Plays ... · G Protein–Coupled Receptor Kinase 2 Plays a Relevant Role in Insulin Resistance and Obesity Lucia Garcia-Guerra,

G Protein–Coupled Receptor Kinase 2 Plays a RelevantRole in Insulin Resistance and ObesityLucia Garcia-Guerra,

1,2Iria Nieto-Vazquez,

1,2Rocio Vila-Bedmar,

1,2María Jurado-Pueyo,

3

Guillermo Zalba,4

Javier Díez,4

Cristina Murga,3

Sonia Fernandez-Veledo,1,2

Federico Mayor Jr.,3

and

Margarita Lorenzo1,2†

OBJECTIVE—Insulin resistance is associated with the patho-genesis of metabolic disorders as type 2 diabetes and obesity.Given the emerging role of signal transduction in these syn-dromes, we set out to explore the possible role that G protein–coupled receptor kinase 2 (GRK2), first identified as a G protein–coupled receptor regulator, could have as a modulator of insulinresponses.

RESEARCH DESIGN AND METHODS—We analyzed the in-fluence of GRK2 levels in insulin signaling in myoblasts andadipocytes with experimentally increased or silenced levels ofGRK2, as well as in GRK2 hemizygous animals expressing 50%lower levels of this kinase in three different models of insulinresistance: tumor necrosis factor-� (TNF-�) infusion, aging, andhigh-fat diet (HFD). Glucose transport, whole-body glucose andinsulin tolerance, the activation status of insulin pathway com-ponents, and the circulating levels of important mediators weremeasured. The development of obesity and adipocyte size withage and HFD was analyzed.

RESULTS—Altering GRK2 levels markedly modifies insulin-mediated signaling in cultured adipocytes and myocytes. GRK2levels are increased by �2-fold in muscle and adipose tissue inthe animal models tested, as well as in lymphocytes frommetabolic syndrome patients. In contrast, hemizygous GRK2mice show enhanced insulin sensitivity and do not developinsulin resistance by TNF-�, aging, or HFD. Furthermore, re-duced GRK2 levels induce a lean phenotype and decreaseage-related adiposity.

CONCLUSIONS—Overall, our data identify GRK2 as an impor-tant negative regulator of insulin effects, key to the etiopatho-genesis of insulin resistance and obesity, which uncovers thisprotein as a potential therapeutic target in the treatment of thesedisorders. Diabetes 59:2407–2417, 2010

Insulin resistance, a diminished ability of cells torespond to the action of insulin, is a key featureassociated with the pathogenesis of metabolic dis-orders such as type 2 diabetes and obesity (1).

Alterations in any of the key components of the insulin-signaling cascade, including negative regulators, havebeen proposed to contribute to insulin resistance (1,2).However, the origin and precise mechanisms mediatinginsulin resistance in physiopathological conditions are notfully understood (3).

Both aging and obesity are associated with increasedrisk of developing type 2 diabetes and cardiovasculardisease. An increase in proinflammatory and a decrease inanti-inflammatory factors is found in the obese state andmay influence glucose homeostasis and insulin sensitivity(4,5). Peripheral tissues exposed to these proinflammatorycytokines develop an insulin-resistant state (6). In fact,obesity is now being considered a chronic state of low-intensity inflammation. In this regard, the cytokine tumornecrosis factor-� (TNF-�) is highly expressed in adiposetissue of obese animals and humans, and obese micelacking either TNF-� or its receptors show protectionagainst developing insulin resistance. The molecularmechanisms underlying TNF-�–mediated insulin resis-tance have been studied in models of murine and humanmyocytes and adipocytes and in vivo (7–11).

Insulin suppresses hepatic glucose production and reg-ulates glucose uptake in muscle and fat through translo-cation of GLUT4 to the cell surface (12,13). Insulin-induced GLUT4 translocation requires at least two signals,one mediated through phosphatidylinositol 3-kinase(PI3K) and another via G�q/11 (14) in 3T3L1 adipocytes.The activated insulin receptor can phosphorylate the Gprotein subunit G�q/11, leading to activation of cdc42 andPI3K, which triggers glucose transport stimulation (14–16). Signaling of receptors via G proteins is regulated by Gprotein–coupled receptor kinases (GRKs), a family ofseven serine/threonine protein kinases that specificallyrecognize and phosphorylate agonist-activated G protein–coupled receptors (GPCRs). This recruits arrestin proteinsthat uncouple receptors from G proteins and promoteinternalization. The ubiquitous GRK2 isoform has beenreported to regulate other pathways independently of itsGPCR phosphorylation ability (17,18). GRK2 can act as aninhibitor of insulin-mediated glucose transport stimulationin 3T3L1 adipocytes by interacting with G�q/11 functionindependently of its kinase activity (19). GRK2 also inhib-its basal and insulin-stimulated glycogen synthesis inmouse liver FL83B cells (20). In this context, we haveinvestigated whether GRK2 may play a relevant physiolog-ical role in the modulation of insulin responses in vivo.

From the 1Department of Biochemistry and Molecular Biology II, Faculty ofPharmacy, Complutense University, Madrid, Spain; the 2CIBER de Diabetesy Enfermedades Metabolicas (CIBERDEM), Madrid, Spain; the 3Departa-mento de Biología Molecular and Centro de Biología Molecular SeveroOchoa (CSIC-UAM) and Instituto de Investigacion Sanitaria Princesa,Madrid, Spain; and the 4Division of Cardiovascular Sciences, Center forApplied Medical Research, University of Navarra, Pamplona, Spain.

Corresponding authors: Cristina Murga, [email protected], and Sonia Fernan-dez-Veledo, [email protected].

Received 4 June 2010 and accepted 3 July 2010. Published ahead of print athttp://diabetes.diabetesjournals.org on 13 July 2010. DOI: 10.2337/db10-0771.

L.G.-G. and I.N.-V. contributed equally to this work.†Deceased.© 2010 by the American Diabetes Association. Readers may use this article as

long as the work is properly cited, the use is educational and not for profit,and the work is not altered. See http://creativecommons.org/licenses/by-nc-nd/3.0/ for details.

The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore be hereby marked “advertisement” in accordance

with 18 U.S.C. Section 1734 solely to indicate this fact.

ORIGINAL ARTICLE

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GRK2 expression is increased in key tissues in differentexperimental models of insulin resistance, and a 50%downregulation of GRK2 levels in hemizygous GRK2�/�

mice is sufficient to protect against TNF-�, aging, orhigh-fat diet (HFD)–induced alterations in glucose ho-meostasis and insulin signaling, strongly arguing for a keyrole for GRK2 in the modulation of insulin sensitivity inphysiological and pathological conditions.

RESEARCH DESIGN AND METHODS

Cell culture and transfections. The human liposarcoma cell line LiSa-2 (21)was used as a cellular model of visceral human adipocytes and was main-tained in Dulbecco‘s modified Eagle‘s medium (DMEM) supplemented with10% fetal serum and antibiotics, at 37°C and 5% CO2. To induce insulinresistance, human adipocytes were cultured for 21 days in serum-freeDMEM/F12 (1:1) as previously described (22). Peripheral blood mononuclearcells, were isolated from blood samples with Lymphoprep (�99% werelymphocytes and monocytes) as previously described (23). The studiesinvolving samples from patients were carried out in accordance with theHelsinki Declaration, and the protocol was approved by the Ethical Commit-tee of the University Clinic of Navarra (supplementary information, availablein an online appendix at http://diabetes.diabetesjournals.org/cgi/content/full/db10-0771/DC1).

Mouse C2C12 myocytes (American Type Culture Collection) and immor-talized murine white adipocytes were maintained in DMEM supplementedwith 10% fetal serum and antibiotics, at 37°C and 5% CO2. Cells weretransiently transfected according to the LipofectAMINE protocol (Invitrogen,Paisley, U.K.), with 4 �g of pcDNA3-GRK2-neo, pcDNA3-GRK2-K220R-hygro,or ad-shGRK2-RNAi, to generate cell lines with increased or decreased GRK2expression, respectively, or with empty vector (pcDNA3-hygro) as a control.Metabolic assays. Glucose tolerance test (GTT) and insulin tolerance test(ITT) were performed as we previously described (24). Glucose concentration(mg/dl) was determined in tail blood samples using an automatic analyzer(Accucheck from Roche). Other measurements were performed from bloodcollected from the tail and centrifuged at 2,500g for 15 min to obtain theserum. Insulin and leptin concentrations were assayed using a sensitive ELISAkit (Linco Research, St. Louis, MO). Triglycerides released were assayed by anenzymatic method using Free Glycerol Determination kit (Sigma-Aldrich, St.Louis, MO).Glucose uptake assay. Cells were stimulated for 30 min with insulin, andglucose uptake was measured during the last 10 min of culture by incorpora-tion of 2-deoxy-D[1–3H]-glucose as previously described (22,24). Individualvalues were expressed as pmol of glucose per 10 min per mg of protein, andresults were expressed as percentage of stimulation over basal (control �100).In vivo signaling assay. Male mice (12- to 14-week-old) were subjected toanesthesia (intraperitoneal injection of 60 mg/kg ketamine per 5 mg/kgxylazine), and 25 mg epididymal adipose tissue was removed from eachmouse. Insulin (1 IU/kg body wt) was then injected via the cava vein, and asimilar amount of epididymal adipose tissue was removed, after 1, 3, and 7min. Tissue samples were immediately frozen in liquid nitrogen and stored at�80°C. Epididymal adipose tissues were homogenized with a Polytronhomogenizer in lysis buffer and centrifuged at 100,000g for 15 min. Thesupernatants were collected, assayed for protein concentration, and stored at�80°C until used. Other methods were performed as specified in the supple-mentary information found in an online appendix.

RESULTS

Insulin signaling is regulated by GRK2 levels in adi-pocytes and myocytes. To study the modulation ofinsulin signaling by GRK2 in relevant insulin-targeted celltypes, we used murine white adipocytic and myocytic celllines expressing enhanced or decreased GRK2 proteinlevels, or overexpressing a catalytically inactive mutant ofGRK2 (Fig. 1A). The increase in glucose uptake elicited byinsulin in both cell types was blocked in the presence ofenhanced GRK2 levels, whereas GRK2 knockdown led to aslightly higher response (Fig. 1B). After acute insulinchallenge, IRS1 tyrosine phosphorylation and AKT activa-tion were impaired upon GRK2 or GRK2-K220R overex-pression, both in adipocytes and in myoblasts (Fig. 1C andD), whereas reduced levels of GRK2 promoted the oppo-

site effect. Basal IRS1 protein levels were higher in cellswith silenced GRK2 and decreased in cells with elevatedGRK2, suggesting a functional relationship between GRK2and IRS1 (Fig. 1C and D). Co-immunoprecipitation exper-iments in adipocytes showed that both endogenous GRK2and IRS1 proteins form a complex under basal conditionsthat is completely disrupted after 10 min of insulin treat-ment (Fig. 2A). GRK2 overexpression enhances theamount of IRS1 associated to the kinase by �87% andprevents the complete disruption of the complex by insu-lin. In contrast, GRK2 downregulation decreases its basalassociation with IRS1 and facilitates a complete disruptionof this complex by insulin.

Interestingly, a similar picture emerges when investigat-ing such parameters in adipose tissue from wild-type (Wt)or GRK2 hemizygous mice, expressing some 50% of GRK2protein (25,26). The amount of basal IRS1/GRK2 com-plexes also depends on GRK2 levels in vivo and insulininfusion disrupts such complexes (Fig. 2B). Also, a fasterkinetics in insulin-mediated IRS1 and AKT phosphoryla-tion (Fig. 2C) is observed in GRK2�/� mice.

Overall, our data indicate that GRK2 levels regulateinsulin signaling in skeletal muscle and adipose tissue bothin cultured cells and in vivo, most probably by mecha-nisms independent of kinase activity and involving theformation of dynamic GRK2/IRS1 complexes.GRK2 protein expression is enhanced under insulin-resistant conditions. If GRK2 is to play a role in themodulation of insulin sensitivity in vivo, the expression ofthis kinase should be altered in conditions characterizedby insulin resistance. Obesity-associated metabolic disor-ders such as type 2 diabetes and insulin resistance can beinduced by TNF-� infusion, aging, or HFD (1). Therefore,we compared GRK2 expression in epididymal white adi-pose tissue (eWAT), liver, and skeletal muscle obtainedfrom 3- and 9-month-old mice, as well as from mice fed ona HFD or treated with TNF-�, using only male mice (Fig.3A and supplementary Table 1, available in an onlineappendix). GRK2 expression levels were enhanced inadipose tissue (from 1.3- to 1.9-fold) and muscle (1.4- to1.7-fold) in every experimental condition studied, andchanges in liver expression were observed upon HFDtreatment. GRK2 expression was also enhanced by �2-fold in eWAT tissue of the ob/ob model of obese mice (Fig.3B) and by 2.5-fold in human adipocytes forced to developan insulin-resistant state upon a chronic challenge withinsulin (Fig. 3C). Finally, because lymphocyte GRK2 levelshave been reported to mirror changes in the kinaseexpression in other organs under several physiopatholog-ical circumstances (27), we analyzed the levels of GRK2 inperipheral blood mononuclear cells obtained from pa-tients with diverse degrees of insulin resistance (supple-mentary Table 2, available in an online appendix). In thesecells, GRK2 levels were higher in insulin-resistant patientscompared with control individuals (Fig. 3D). Althoughpreliminary, our results indicate that GRK2 levels arealtered in murine experimental models and in humansamples under insulin-resistant conditions, suggesting thatthis kinase might mediate the ability to induce insulinresistance of different neurohumoral factors that are al-tered in these situations (28,29).GRK2�/� mice do not develop TNF-�–induced insulinresistance. To test the hypothesis that such moderatechanges in GRK2 expression levels may indeed alterinsulin signaling in vivo, we studied whole-body insulinaction and glucose homeostasis in age-matched wild-type

CONTROL OF INSULIN RESISTANCE AND OBESITY BY GRK2

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and GRK2�/� male mice. These haploinsufficient miceconstitute a good model to recapitulate differences inGRK2 expression in the same range to those observed inpathological conditions, as well as to evaluate the poten-tial of therapeutic strategies aimed at decreasing GRK2functionality in vivo. Whereas GRK2 knock-out mice die inutero with a marked cardiac hypoplasia (30) and thecardiac phenotype of GRK2�/� mice has been well char-acterized (31,32), insulin signaling has not been investi-gated so far in these animals.

Because TNF-� infusion induces insulin resistance inmurine models in vivo (24,33), we examined insulin sen-sitivity by GTTs and ITTs in wild-type and GRK2�/�

3-month-old male mice after a 48-h treatment with TNF-�(Fig. 4A and C). In wild-type but not in GRK2�/� mice,TNF-� produced a significant 29% increase in fasting bloodglucose levels (Table 1). Moreover, the additional pro-nounced hyperglycemic effect of TNF-� after glucoseinjection observed in wild-type mice was attenuated inGRK2�/� mice (Fig. 4A). The improved glucose toleranceobserved was not due to an increase in circulating insulinbecause GRK2�/� mice had lower circulating insulin levelsthan wild-type mice in both the fasted state (Table 1) and

during the GTT (Fig. 4B). These results suggest that themore efficient glucose clearance observed in GRK2�/�

mice is caused by enhanced peripheral insulin sensitivity.In line with this notion, the hypoglycemic effect of insulininjection was impaired upon TNF-� treatment in wild-typeanimals but not in GRK2�/� mice (Fig. 4C).

Wild-type mice treated with TNF-� showed a completeimpairment in insulin-induced AKT phosphorylation inskeletal muscle, adipose tissue, and liver (Fig. 4D andsupplementary Fig. S2, available in an online appendix,and supplementary Table 1) that was not detected inGRK2�/� mice. Interestingly, TNF-� substantially in-creased GRK2 protein content in eWAT and muscle ofwild-type mice (by 82 and 42%, respectively) (Fig. 4D,supplementary Fig. S2, and supplementary Table 1),whereas the amount of GRK2 remained unaltered (in liverand muscle) or below basal wild-type levels (adipose)upon TNF-� treatment in GRK2�/� mice (Fig. 4D, supple-mentary Fig S2, and supplementary Table 1). Altogether,these results suggest that GRK2 is a negative modulator ofinsulin sensitivity and that lower GRK2 levels protectagainst the systemic insulin resistance induced by TNF-�.

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FIG. 1. Modulation on insulin sensitivity by GRK2. Immortalized murine white adipocytes and C2C12 myocytes were transiently transfected with emptyvector (control, C), pcDNA3-GRK2-neo (�), the catalytically inactive pcDNA3-GRK2–K220R (K), or shGRK2-RNAi (�) to upregulate or knock-downGRK2 expression, respectively. A: Lysates were analyzed by Western blot with the corresponding antibodies against GRK2 and �-actin. For examplesof full-length blots, see supplementary Fig. S3, available in an online appendix. B: Adipocytes and myocytes were cultured for 24 h in serum-free andlow-glucose medium and stimulated in the absence or presence of 10 nmol/l insulin for 30 min. Glucose uptake was measured during the last 10 min byincorporation of 2-deoxyglucose into the cells. Results were expressed as percentage of stimulation over basal of control cells (adipocytes, 5.3 � 0.3pmol glucose/mg protein/10 min; myocytes, 45.8 � 3.2 pmol glucose/mg protein/10 min) and are means � SEM of five independent experiments. C:Adipocytic and (D) myocytic cell lines were serum-starved overnight and then incubated in the absence or presence of 10 nmol/l insulin (Ins) for 10min. Lysates were analyzed by Western blot with the corresponding antibodies against total and/or phosphorylated forms of IRS1 (Tyr 608), AKT(Ser473), ERK1/2 (Thr202/Tyr204), GRK2, and �-actin. Representative immunoblots of 4–6 independent experiments and densitometric analysis areshown in (A, C, and D). *P < 0.01. For examples of full-length blots, see supplementary Fig. S3. Data in A, C, and D were normalized with the indicatedcontrols, and results are expressed as percent over basal (control cells transfected with empty vector).

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GRK2 downregulation prevents the development ofaging-associated insulin resistance. In the model ofaging-associated insulin resistance, GRK2 downregulation

caused no differences in the GTTs or ITTs of young(3-month-old) male mice (Fig. 5A–C), whereas in adult9-month-old GRK2�/� male mice, glucose tolerance was

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FIG. 2. Modulation of insulin signaling by GRK2 levels involves the formation of dynamic GRK2/IRS1 complexes. Immortalized murine whiteadipocytes were transiently transfected with empty vector (control, C), pcDNA3-GRK2-neo (�), or shGRK2-RNAi (�) to upregulate orknock-down GRK2 expression, respectively. A: Adipocytic cell lines were serum-starved overnight and then incubated in the absence or presenceof 10 nmol/l insulin for 10 min. Total protein from adipocytic cells (1 mg) was immunoprecipitated with the anti-IRS1 or anti-IgG antibodies, andthe resulting immune complexes were analyzed by Western blot with the corresponding antibodies against GRK2 and IRS1. B: Samples of eWATwere obtained at the indicated times from Wt and GRK2�/� adult male mice undergoing insulin (Ins) infusion (1 IU/kg body wt) as detailed inRESEARCH DESIGN AND METHODS. Lysates were immunoprecipitated with an anti-IRS1 antibody, and the resulting immune complexes were analyzedby Western blot with the corresponding antibodies against GRK2 and IRS1. C: The same lysates were analyzed by Western blot with thecorresponding antibodies against total and/or phosphorylated forms of IRS1 (Tyr 608), AKT (Ser473), and �-actin. Representative immunoblotsof 4–6 independent experiments and densitometric analysis are shown. Data were normalized with the indicated controls, and results obtainedin nonstimulated control cells (A) or Wt animals (B, C) were taken as 100%. *P < 0.01.

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FIG. 3. GRK2 protein expression is enhanced under insulin-resistance conditions. A: eWAT, liver, and muscle from 3- or 9-month-old male mice,fed on a HFD for 12 weeks or treated with TNF-� (0.1 �g/kg body wt) for 48 h; (B) eWAT from Wt and ob/ob mice; (C) insulin-sensitive andinsulin-resistant human adipocytes; and (D) peripheral blood mononuclear cells (PBMCs) collected from blood samples of insulin-sensitiveindividuals and insulin-resistant patients (supplementary Table 2) were lysed and subjected to immunoblot analysis with antibodies againstGRK2 and �-actin as a loading control. Data in A, B, and C are means � SEM of 4 independent experiments. Data in A were normalized with theindicated control, and results are expressed as percent over basal (3 month, Wt mice). Results in D represent the means � SEM of 10 controlsubjects and 25 metabolic syndrome patients. Representative blots are shown. *P < 0.01.



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improved compared with wild-type (Fig. 5D). A similartendency had already been observed at 5 months of age inGRK2�/� mice (supplementary Fig. S1, available in anonline appendix). Aging also induced a 57% increase infasting glucose concentration in wild-type mice, limited to29% in GRK2�/� mice (Table 1). Circulating levels ofinsulin were higher in 9-month-old wild-type than inGRK2�/� mice in response to an identical glucose load(Fig. 5E). Consistently, GRK2�/� mice exhibited an en-hanced decline in blood glucose levels upon administra-tion of insulin (Fig. 5F) and lower fasting serum insulinlevels (Table 1). Altogether, these results suggest in-creased whole-body insulin sensitivity in GRK2�/� mice, inagreement with the values obtained for the homeostasismodel assessment index (Table 1).

Insulin-stimulated AKT phosphorylation was markedlyimpaired with age in eWAT, liver, and muscle in wild-typemice, while GRK2�/� animals maintained a robust activa-tion of this pathway (Fig. 5G, supplementary Fig. S2, andsupplementary Table 1). Interestingly, in adult animals,GRK2 protein expression was enhanced by 33 and 68% ineWAT and muscle, respectively, compared with youngmice (Figs. 3A and 5G, supplementary Fig. S2, and supple-mentary Table 1), while GRK2 levels in adult GRK2�/�

mice were below the expression detected at 3 months ofage in wild-type animals in all tissues examined (Fig. 5G,supplementary Fig. S2, and supplementary Table 1). Over-all, these data suggest there is a threshold above which theenhanced GRK2 expression observed with age markedlyaffects insulin sensitivity in vivo.GRK2�/� mice display protection against HFD-inducedinsulin resistance and obesity. After a HFD regime for12 weeks, wild-type mice gained significantly more weight

than GRK2�/� mice (Fig. 6A). The overall increase in bodyweight was 67% in wild-type and only 53% in GRK2�/�

mice (Fig. 6B), without changes in food intake (Fig. 6C).Consistently, even when adipocyte diameter was in-creased in both groups, adiposity in GRK2 hemizygousmice did not go beyond a certain threshold that wasexceeded only in wild-type mice, with the size of HFD-fedGRK2�/� epididymal adipocytes being 45% inferior to thatof wild-type mice (Fig. 6D).

GTT analysis indicated a clear deterioration in glucosemetabolism in wild-type but not in HFD-fed GRK2�/�

mice. In the HFD-fed state, GRK2�/� mice did not developglucose intolerance (Fig. 6E), because we detect no sta-tistically significant changes in glucose area between chowdiet (CD)-fed and HFD-fed GRK2�/� mice, which showedlower blood glucose levels (Table 1) and an enhancedreduction in blood glucose induced by insulin (Fig. 6F).HFD caused a marked increase in circulating levels ofglucose, insulin, and triglycerides after 12 weeks of feed-ing (Table 1) in wild-type mice, whereas these changeswere much more modest in GRK2�/� animals. Theseparameters were not altered with CD in either type ofanimal (data not shown). In summary, HFD promoted amarked insulin-resistance state in wild-type mice, in ac-cordance with the observed increase in homeostasismodel assessment values (Table 1), whereas insulin sen-sitivity was preserved in GRK2�/� mice.

Insulin-induced AKT phosphorylation was dramaticallydecreased in adipose tissue, liver, and muscle of wild-typebut not HFD-fed GRK2�/� mice (Fig. 6G, supplementaryFig. S2, and supplementary Table 1). On the other hand, inagreement with data in Fig. 3A, GRK2 protein levels weresignificantly increased in eWAT (84%), liver (26%), and

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FIG. 4. GRK2�/� mice do not develop TNF-�–induced insulin resistance. Wt (open symbols, solid lines) and GRK2�/� (filled symbols, dotted lines)adult male mice were treated with TNF-� (0.1 �g/g body wt, circles) or vehicle (100 �l PBS plus 0.1% BSA, squares) for 48 h. A: GTTs wereperformed on animals that had been fasted for 24 h and were given an injection of glucose (2 g/kg body wt). B: Circulating insulin levels duringthe GTTs. C: ITTs were performed on fed male mice that had received insulin (0.8 IU/kg body wt). Glucose concentration was determined in bloodsamples obtained from the tail vein. Results are means � SEM of eight animals for each group. Histograms on the right show the incremental areaunder the curve. *P < 0.01. D: Wt and GRK2�/� male mice were treated or not with insulin (1 IU/kg body wt) for 15 min, and eWAT, liver, andmuscle were removed. Lysates were subjected to Western blot with the indicated antibodies against total and phosphorylated AKT (Ser473),GRK2, and �-actin. Representative immunoblots of four independent experiments are shown. See supplementary Fig. S2 and supplementary Table1 for detailed quantification of these data. For examples of full-length blots, see supplementary Fig. S4, available in an online appendix.



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muscle (63%) of wild-type but not GRK2�/� mice fed on aHFD (Fig. 6G, supplementary Fig. S2, and supplementaryTable 1). These results indicate that, even in differentcellular settings, whenever glucose intolerance develops,GRK2�/� mice always maintain the levels of GRK2 proteinbelow those detected in wild-type and thus below a giventhreshold that helps to prevent the development of insulininsensitivity.GRK2�/� mice exhibit reduced adiposity. In addition tothe data shown in Fig. 6A–C, a detailed weekly analysis ofthe weight of wild-type and GRK2�/� animals from wean-ing to 15 months of age (Fig. 7A) revealed less weight gainfor both male and female GRK2�/� mice (Fig. 7B), withoutdifferences in daily food intake (Fig. 7C). Body weightdifferences were detected as early as at 5 months of age(Fig. 7A), and at 9 months of age, when some wild-typemice are visibly obese (Fig. 7D), the average body weightgain was 65 and 56% in male and female wild-type mice,respectively, but only 56 and 43% in GRK2�/� mice (Fig.7B). Body fat distribution was studied by magnetic nuclearresonance imaging (MNRI) (Fig. 7E), showing that9-month-old GRK2�/� mice had 33% less overall fat andsmaller fat depots than age-matched wild-type littermates(Fig. 7F). Epididymal fat mass was reduced by 30% inGRK2�/� compared with wild-type mice at 9 months of age(Fig. 7G), and GRK2�/� mice exhibited reduced adipocyticsize in epididymal (threefold), retroperitoneal (4.6-fold),and inguinal (threefold) fat (Fig. 7H). Interestingly, adecrease in circulating leptin levels was also detected inGRK2�/� animals at 9 months of age (Table 1). Theseresults indicate that downregulation of GRK2 is sufficientto induce a lean phenotype, which uncovers a previouslyunidentified role for GRK2 in the regulation of adiposity.

DISCUSSION

In this report, we uncover GRK2 as a key modulator ofinsulin sensitivity in vivo. In cultured adipocytes andmyoblasts, increased GRK2 or GRK2-K220R levels inhibitinsulin-stimulated glucose uptake and signaling in a ki-nase-activity independent manner, by mechanisms involv-ing the formation of dynamic GRK2/IRS1 complexes.GRK2 expression is enhanced by �2-fold in insulin-resis-tant human adipocytes, in blood mononuclear cells frominsulin-resistant patients, and in adipose and muscle tis-sues in either TNF-�–, aging-, or HFD-induced insulin-resistance models. Importantly, GRK2�/� mice maintainglucose tolerance and insulin signaling in the major insu-lin-responsive tissues under such experimental conditions,suggesting that enhanced GRK2 expression above a cer-tain threshold markedly impairs insulin sensitivity in vivo.Finally, we find that GRK2 levels also regulate adiposityduring aging. Because GRK2 haploinsufficient animalsshow a phenotype quite similar to mice deficient in otherwell established negative regulators of insulin signalingsuch as PTP1B (24,33,34), GRK2 can be regarded as a bonafide novel physiological regulator of insulin action andleanness.

Previous reports have indicated that GRK2 could act asan inhibitor of insulin action in cellular models. Binding ofGRK2 to G�q/11 impairs insulin-stimulated glucose uptakein 3T3L1 preadipocytes (19), and enhanced GRK2 levelsfavor insulin resistance induced by endothelin-1 in 3T3L1cells (29) or by chronic �-adrenergic receptor stimulationin HEK-293 cells (28). GPCR agonists promote GRK2interaction with G�q and also with IRS1, resulting inT

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decreased insulin-stimulated glucose transport, IRS-serinephosphorylation, and IRS1 degradation (29). Our data incultured cells as well as in adipose tissue in vivo show thatIRS1 levels as well as the amount of basal GRK2-IRS1complexes depend on GRK2 expression and that insulinstimulation rapidly disrupts basal IRS1/GRK2 complexes,which is hampered in the presence of elevated levels ofGRK2. Overall, these data suggest that altered GRK2 levelscould lead to modulation of insulin signals through GRK2-G�q/11 binding, GRK2-IRS1 association, and altered endo-thelin-1 or �-adrenergic-mediated transmodulation of theinsulin pathway. The precise contribution of such mecha-nisms in different physiological conditions remains to beinvestigated.

Interestingly, �-arrestin2, another member of the GPCRsignal transduction pathway, positively regulates insulinsensitivity by serving as a scaffold of AKT and Src to theinsulin receptor (35). �-arrestin2 levels are downregulatedin liver and muscle in animal models and patients withinsulin resistance. Therefore, simultaneous upregulationof GRK2 and downregulation of �-arrestin2 in insulin-resistant conditions could lead to major alterations in theinsulin signaling pathways and in GPCR–insulin receptor

cross-talk. It is worth noting that elevated levels of insulinand IGF-1 have been shown to exert differential effects onGRK2 and �-arrestin2 expression (36,37). Enhanced GRK2levels could alter the proposed novel insulin receptor–arrestin signaling pathway by redirecting arrestin to otherinteractors, such as GPCR, or, given the reported directinteractions of the kinase with AKT or Src, by inhibitingthe formation of the arrestin/AKT/Src complexes.

GRK2 is degraded by the proteasome pathway by asso-ciating with the Mdm2 E3-ubiquitin ligase, and activationof the PI3K/AKT pathway by IGF-1 blocks Mdm2-mediatedGRK2 degradation, leading to enhanced GRK2 levels (38).Consistently, chronic insulin has been recently shown toincrease GRK2 levels in HEK-293 and liver FL83B cells(20,28), which is in agreement with the upregulation ofGRK2 we observe after insulin treatment in human adipo-cytic cells. Thus, it is tempting to suggest that the hyper-insulinemia associated with aging-, HFD-, or TNF-�–induced insulin-resistant conditions, and clinicallyassociated with obesity and type 2 diabetes, would triggerthe observed upregulation of GRK2 in such conditions.Consistent with our findings, enhanced GRK2 expression

FIG. 5. GRK2 downregulation prevents the development of aging-associated insulin resistance. A: GTTs were performed on Wt (open circles, solidlines) and GRK2�/� (filled circles, dotted lines) 3-month-old male mice as in Fig. 4A. B: Circulating insulin levels during the GTTs. C: ITTs wereperformed on Wt (open circles) and GRK2�/� (filled circles) 3-month-old male mice as in Fig. 4C. D–F: GTTs and ITTs were performed as inprevious panels in Wt (open circles) and GRK2�/� (filled circles) 9-month-old male mice. *P < 0.01. G: Lysates from Wt and GRK2�/� 3- and9-month-old male mice treated or not with insulin were processed as in Fig. 4D. Representative immunoblots of four independent experimentsare shown. See supplementary Fig. S2 and supplementary Table 1 for detailed quantification of these data.



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has been reported in tissues from obese Zucker rats, amodel of insulin resistance and hyperinsulinemia (39).

An altered cytokine expression pattern typical of insu-lin-resistant states could also contribute to enhance GRK2expression levels. TNF-� or IL-6 increase the expression ofother negative modulators of the insulin signaling cascadelike PTP1B (24,40), and we find that TNF-� also enhancesGRK2 protein content in adipose tissue and muscle ofwild-type mice in the presence of mild hyperinsulinemia.As in GRK2�/� mice, PTP1B-deficient animals also exhibitprotection against insulin resistance induced by TNF-�(24) or IL-6 (40). Proinflammatory mediators can modulateendogenous GRK2 expression in a cell-type specific fash-ion (41), and an increase in GRK2 protein is found uponchronic IL-1� treatment (26,42). Interestingly, GRK2 ap-pears to have a relevant role in the etiology and/or in the

development of several inflammatory diseases such asmultiple sclerosis and autoimmune arthritis (43). A rolefor GRK2 in states of insulin resistance associated withinflammatory diseases deserves further investigation.

The fact that the 50% downregulation of GRK2 levels issufficient per se to protect against TNF-�-, aging-, orHFD-induced alterations in glucose homeostasis and insu-lin signaling strongly supports that this kinase is a keymodulator of insulin sensitivity in vivo and suggests newtherapeutic strategies against type 2 diabetes and obesity.In fact, in animal models of type 2 diabetes, GRK2 inhibi-tion through systemic delivery of small peptides derivedfrom its catalytic domain results in improved glucosehomeostasis (28,44). It remains to be established whetherinhibition of catalytic activity, downregulation of proteinexpression, or targeted disruption of the specific interac-

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FIG. 6. GRK2�/� mice display protection against diet-induced obesity. Wt (open symbols, solid lines) and GRK2�/� (filled symbols, dotted lines)male mice were fed a CD or HFD for 12 weeks. A: Body weight evolution and (B) percentage of change in body weight during the 12 weeks oftreatment. C: Daily food intake on CD or HFD. D: Paraffin-embedded sections of epididymal fat pads from Wt and GRK2�/� male mice fed on CDor HFD and stained with hematoxylin and eosin (magnification �10; scale bar 50 �m for all pictures). Relative adipocyte size was calculated asdetailed in RESEARCH DESIGN AND METHODS. Data are means � SEM of 10–20 animals per group. E: GTTs (circles, HFD; squares, CD) and (F) ITTswere performed as in Fig. 4A and C, respectively. *P < 0.01. G: Lysates from Wt and GRK2�/� male mice fed CD or HFD for 12 weeks treated ornot with insulin were processed as in Fig. 4D. Representative immunoblots of four independent experiments are shown. See supplementary Fig.S2 and supplementary Table 1 for detailed quantification of these data. (A high-quality color representation of this figure is available in the onlineissue.)



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tion of GRK2 with insulin signaling pathway componentsare the more appropriate strategies for specifically improv-ing insulin sensitivity.

The GRK2�/� mice constitute a good model that reca-pitulates a sustained systemic inhibition of kinase func-tionality. We report here that GRK2 levels are importantmodulators of age- and diet-induced adiposity. GRK2�/�

mice gained less weight and showed diminished adipocytesize with a HFD, and adult hemizygous animals displayed

reduced adiposity and lower circulating levels of insulinand leptin. Aging is associated with fat mass accretion andwith decreased peripheral insulin sensitivity in humansand rodents (45), and impaired insulin actions in adiposetissue could represent a key step leading to the overallinsulin-resistance characteristic of adult and/or obese an-imals (46). Adipocyte-conditioned medium impairs insulinsignaling in muscle cells (22) and hepatocytes (47), andmice that exhibit reduced adiposity typically display im-

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mice stained with hematoxylin and eosin. (Magnification �10; scale bar 50 �m for all pictures.) Relative adipocyte size was calculated as in Fig.6D. Data are means � SEM of 5–30 animals per group. *P < 0.01. (A high-quality color representation of this figure is available in the onlineissue.)



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proved glucose tolerance and increased insulin sensitivity(46). Therefore, it is possible that part of the insulinhypersensitivity detected in GRK2�/� mice and the protec-tion afforded to the development of insulin-resistant con-ditions in these animals could be related to the observedchanges in adiposity. Future experiments involving tissue-specific downregulation will provide new insight into theunderstanding of the role of GRK2 in obesity and insulinresistance.

ACKNOWLEDGMENTS

This work was supported by Grants BFU2008-04043 (toM.L.), SAF2008-0552 (to F.M. Jr.), and SAF2007-62553 (toG.Z.) from Ministerio de Ciencia e Innovacion, Spain,S-SAL-0159-2006 from Comunidad de Madrid, Spain (toM.L. and F.M. Jr.), Fundacion Ramon Areces (to F.M. Jr.),the European Union (InGenious HyperCare, grant LSHM-CT-2006-037093 to J.D., through the agreement betweenthe Foundation for Applied Medical Research [FIMA] and“UTE project CIMA”), The Cardiovascular Network (RE-CAVA, RD06-0014/0037 and 0014/008 to F.M. Jr. and J.D.,respectively), CIBER de Diabetes y Enfermedades Meta-bolicas Asociadas, and PS09/01208 (to C.M.) from Minis-terio Sanidad y Consumo-Instituto Carlos III, Spain. Wealso acknowledge the support of COST Action BM0602from the European Commission (to M.L.). No potentialconflicts of interest relevant to this article were reported.

L.G.-G. and R.V.-B. researched data and contributed tothe discussion. I.N.-V. wrote the manuscript, researcheddata, and contributed to the discussion. M.J.-P. researcheddata. G.Z. and J.D. collected samples and reviewed/editedthe manuscript. C.M., F.M. Jr., M.L., and S.F.-V. contrib-uted to the discussion and wrote/edited the manuscript.

The authors thank Dr. Oscar Beloqui from the Univer-sity Clinic of Navarra for the recollection of clinical data ofstudied patients.

This work is dedicated to the memory of Prof. MargaritaLorenzo, who passed away April 7, 2010, at the age of 51.

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ORIGINAL ARTICLE G Protein–Coupled Receptor Kinase 2 Plays ... · G Protein–Coupled Receptor Kinase 2 Plays a Relevant Role in Insulin Resistance and Obesity Lucia Garcia-Guerra,