g protein-coupled receptor kinase 2...

G PROTEIN-COUPLED RECEPTOR KINASE 2 (GRK2) PLAYS A RELEVANT ROLE

IN INSULIN RESISTANCE AND OBESITY

Lucia Garcia-Guerra 1, 2

, Iria Nieto-Vazquez1, 2

, Rocio Vila-Bedmar1, 2

, María Jurado-Pueyo3,

Guillermo Zalba4, Javier Díez

4, Cristina Murga

3, *, Sonia Fernández-Veledo

1,2, *, Federico

Mayor Jr. 3

and Margarita Lorenzo1,2,+

.

1Department of Biochemistry and Molecular Biology II, Faculty of Pharmacy, Complutense

University; 28040 Madrid (Spain); 2CIBER de Diabetes y Enfermedades Metabólicas

(CIBERDEM), 3Departamento de Biología Molecular and Centro de Biología Molecular Severo

Ochoa (CSIC-UAM) and Instituto de Investigación Sanitaria Princesa, 28049 Madrid (Spain), 4Division of Cardiovascular Sciences, Center for Applied Medical Research, University of

Navarra, 31008 Pamplona, Spain.

L. G-G. and I. N-V. contributed equally to this work

Corresponding authors.

Cristina Murga

[email protected]

Sonia Fernández-Veledo

[email protected]

Additional information for this article can be found in an online appendix at

http://diabetes.diabetesjournals.org

Submitted 4 June 2010 and accepted 3 July 2010.

This is an uncopyedited electronic version of an article accepted for publication in Diabetes. The American Diabetes Association, publisher of Diabetes, is not responsible for any errors or omissions in this version of the manuscript or any version derived from it by third parties. The definitive publisher-authenticated

version will be available in a future issue of Diabetes in print and online at http://diabetes.diabetesjournals.org.

Diabetes Publish Ahead of Print, published online July 13, 2010

Copyright American Diabetes Association, Inc., 2010

Control of insulin resistance and obesity by GRK2

Objective - Insulin resistance is associated with the pathogenesis of metabolic disorders as type-

2-diabetes and obesity. Given the emerging role of signal transduction in these syndromes, 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 insulin

responses.

Research Design and Methods - We analyzed the influence of GRK2 levels in insulin signaling

in myoblasts and adipocytes with experimentally increased or silenced levels of GRK2, and in

GRK2 hemizygous animals expressing 50% lower levels of this kinase in three different models

of insulin resistance: TNFα infusion, aging and high fat diet (HFD). Glucose transport, whole

body glucose and insulin tolerance, the activation status of insulin pathway components, and the

circulating levels of important mediators were measured. The development of obesity and

adipocyte size with age and HFD was analyzed.

Results - Altering GRK2 levels markedly modifies insulin-mediated signaling in cultured

adipocytes and myocytes. GRK2 levels are increased by approximately 2-fold in muscle and

adipose tissue in the animal models tested, and in lymphocytes from metabolic syndrome

patients. In contrast, hemizygous GRK2 mice show enhanced insulin sensitivity and do not

develop insulin resistance by TNFα, aging or HFD. Furthermore, reduced GRK2 levels induce a

lean phenotype and decrease age-related adiposity.

Conclusions - Overall, our data identify GRK2 as an important negative regulator of insulin

effects, key to the etiopathogenesis of insulin resistance and obesity, what uncovers this protein

as a potential therapeutic target in the treatment of these disorders.

nsulin resistance, a diminished ability of

cells to respond to the action of insulin, is

a key feature associated with the

pathogenesis of metabolic disorders such as

type-2-diabetes and obesity (1). Alterations in

any of the key components of the insulin

signaling cascade, including negative

regulators, have been proposed to contribute

to insulin resistance (1;2). However, the

origin and precise mechanism(s) mediating

insulin resistance in physiopathological

conditions are not fully understood (3).

Both aging and obesity are associated with

increased risk of developing type-2-diabetes

and cardiovascular disease. An increase in

pro-inflammatory and a decrease in anti-

inflammatory factors is found in the obese

state and may influence glucose homeostasis

and insulin sensitivity (4;5). Peripheral tissues

exposed to these pro-inflammatory cytokines

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 Tumor Necrosis Factor

(TNF)α is highly expressed in adipose tissue

of obese animals and humans, and obese mice

lacking either TNFα or its receptors show

protection against developing insulin

resistance. The molecular mechanisms

underlying TNFα-mediated insulin resistance

have been studied in models of murine and

human myocytes and adipocytes and in vivo

(7-11).

Insulin suppresses hepatic glucose production

through translocation of glucose transporter

(GLUT)4 to the cell surface (12;13). Insulin-

induced GLUT4 translocation requires at least

two signals, one mediated through

I


phosphatidylinositol 3-kinase (PI3K) and

another via Gαq/11 (14) in 3T3L1 adipocytes.

The activated Insulin Receptor (IR) can

phosphorylate the G protein subunit Gαq/11,

leading to activation of cdc42 and PI3K, what

triggers glucose transport stimulation (14-16).

Signaling of receptors via G proteins is

regulated by G protein-coupled receptor

kinases (GRKs), a family of seven

serine/threonine protein kinases that

specifically recognize and phosphorylate

agonist-activated G protein-coupled receptors

(GPCRs). This recruits arrestin proteins that

uncouple receptors from G proteins and

promote internalization. The ubiquitous

GRK2 isoform has been reported to regulate

other pathways independently of its GPCR

phosphorylation ability (17;18). GRK2 can

act as an inhibitor of insulin-mediated glucose

transport stimulation in 3T3L1 adipocytes by

interacting with Gαq/11 function

independently of its kinase activity (19).

GRK2 also inhibits basal and insulin-

stimulated glycogen synthesis in mouse liver

FL83B cells (20). In this context, we have

investigated whether GRK2 may play a

relevant physiological role in the modulation

of insulin responses in vivo. GRK2 expression

is increased in key tissues in different

experimental models of insulin resistance and

a 50% down-regulation of GRK2 levels in

hemizygous GRK2+/-

mice is sufficient to

protect against TNFα, aging or high fat diet

(HFD)-induced alterations in glucose

homeostasis and insulin signaling, strongly

arguing for a key role for GRK2 in the

modulation of insulin sensitivity in

physiological 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 maintained in DMEM supplemented

with 10% fetal serum and antibiotics, at 37°C

and 5% CO2. To induce insulin resistance

human adipocytes were cultured for 21 days

in serum-free DMEM/F12 (1:1) as previously

described (22). Peripheral blood mononuclear

cells (PBMCs), were isolated from blood

samples with Lymphoprep (≥99% were

lymphocytes and monocytes) as previously

described (23). The studies involving samples

from patients were carried out in accordance

with the Helsinki Declaration, and the

protocol was approved by the Ethical

Committee of the University Clinic of

Navarra (see Supplemental Information in the

online appendix available at

http://diabetes.diabetesjournals.org).

Mouse C2C12 myocytes (American Type

Culture Collection) and immortalized murine

white adipocytes were maintained in DMEM

supplemented with 10% fetal serum and

antibiotics, at 37°C and 5% CO2. Cells were

transiently transfected according to the

LipofectAMINETM

protocol (Invitrogen,

Paisley, UK), with 4µg of pcDNA3-GRK2-

neo, pcDNA3-GRK2-K220R-hygro or ad-

shGRK2-RNAi, to generate cell lines with

increased or decreased GRK2 expression,

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

blood collected from the tail and centrifuged

at 2,500g for 15 min to obtain the serum.

Insulin and leptin concentrations were

assayed using a sensitive ELISA-kit (Linco

Research, St. Louis, MO). Triglycerides (TG)

released were assayed by an enzymatic

method using Free Glycerol Determination kit

(Sigma-Aldrich, St. Louis, MO).

Glucose Uptake Assay. Cells were

stimulated for 30 min with insulin, and

glucose uptake was measured during the last

10 min of culture by incorporation of 2-


deoxy-D[1-3H]-glucose as previously

described (22;24). Individual values were

expressed as pmol of glucose per 10 min per

mg of protein, and results were expressed as

percentage of stimulation over basal

(control=100).

In vivo signaling assay. Male mice (12-14

week-old) were subjected to anaesthesia

(intraperitoneal injection of 60 mg/kg

ketamine per 5 mg/kg xylazine), and 25 mg

epididymal adipose tissue was removed from

each mouse. Insulin (1 IU/kg body weight)

was then injected via the cava vein and a

similar amount of epididymal adipose tissue

was removed, after 1, 3 and 7 min, Tissue

samples were immediately frozen in liquid

nitrogen and stored at -80°C. Epididymal

adipose tissues were homogenized with a

Polytron homogenizer in lysis buffer and

centrifuged at 100,000g for 15 min. The

supernatants were collected, assayed for

protein concentration, and stored at -80°C

until used.

Other Methods were performed as specified

in Supplemental Information.

RESULTS

Insulin signaling is regulated by GRK2

levels in adipocytes and myocytes. To study

the modulation of insulin signaling by GRK2

in relevant insulin-targeted cell types, we used

murine white adipocytic and myocytic cell

lines expressing enhanced or decreased GRK2

protein levels, or over-expressing a

catalytically inactive mutant of GRK2 (Fig.

1A). The increase in glucose uptake elicited

by insulin in both cell types was blocked in

the presence of enhanced GRK2 levels,

whereas GRK2 knockdown led to a slightly

higher response (Fig. 1B). Following acute

insulin challenge, IRS1 tyrosine

phosphorylation and AKT activation were

impaired upon GRK2 or GRK2-K220R

overexpression both in adipocytes and in

myoblasts (Fig. 1C and 1D), while reduced

levels of GRK2 promoted the opposite effect.

Basal IRS1 protein levels were higher in cells

with silenced GRK2, and decreased in cells

with elevated GRK2, suggesting a functional

relationship between GRK2 and IRS1 (Fig.

1C and 1D). Co-immunoprecipitation

experiments in adipocytes showed that both

endogenous GRK2 and IRS1 proteins form a

complex under basal conditions that is

completely disrupted after 10 minutes of

insulin treatment (Fig. 2A). GRK2 over-

expression enhances the amount of IRS1

associated to the kinase by approximately

87% and prevents the complete disruption of

the complex by insulin. In contrast, GRK2

downregulation decreases its basal association

with IRS1 and facilitates a complete

disruption of this complex by insulin.

Interestingly, a similar picture emerges when

investigating such parameters in adipose

tissue from wild-type (Wt) or GRK2

hemizygous mice, expressing some 50% of

GRK2 protein (25;26). The amount of basal

IRS1/GRK2 complexes also depends on

GRK2 levels in vivo and insulin infusion

disrupts such complexes (Fig. 2B). Also, a

faster kinetics in insulin-mediated IRS1 and

AKT phosphorylation (Fig. 2C) is observed in

GRK2+/-

mice.

Overall, our data indicate that GRK2 levels

regulate insulin signaling in skeletal muscle

and adipose tissue both in cultured cells and

in vivo, most probably by mechanisms

independent of kinase activity and involving

the formation of dynamic GRK2/IRS1

complexes.

GRK2 protein expression is enhanced

under insulin-resistant conditions. If GRK2

is to play a role in the modulation of insulin

sensitivity in vivo, the expression of this

kinase should be altered in conditions

characterized by insulin resistance. Obesity-

associated metabolic disorders such as type-2-

diabetes and insulin resistance can be induced

by TNFα infusion, aging or HFD (1).

Therefore, we compared GRK2 expression in

epididymal white adipose tissue (eWAT),


liver and skeletal muscle obtained from 3 and

9-month-old mice, and from mice fed on a

HFD, or treated with TNFα using only male

mice (Fig. 3A and Suppl. Table 1). GRK2

expression levels were enhanced in adipose

tissue (from 1.3 to 1.9-fold) and muscle (1.4

to 1.7-fold) in every experimental condition

studied, and changes in liver expression were

observed upon HFD treatment. GRK2

expression was also enhanced by more than 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 develop an

insulin-resistant state upon a chronic

challenge with insulin (Fig. 3C). Finally,

since lymphocyte GRK2 levels have been

reported to mirror changes in the kinase

expression in other organs under several

physiopathological circumstances (27), we

analyzed the levels of GRK2 in peripheral

blood mononuclear cells obtained from

patients with diverse degrees of insulin

resistance (Suppl. Table 2). In these cells

GRK2 levels were higher in insulin resistant

patients compared to control individuals (Fig.

3D). Although preliminary, our results

indicate that GRK2 levels are altered in

murine experimental models and in human

samples under insulin resistant conditions,

suggesting that this kinase might mediate the

ability to induce insulin resistance of different

neurohumoral factors that are altered in these

situations (28;29).

GRK2+/-

mice do not develop TNFαααα-induced insulin resistance. To test the hypothesis that

such moderate changes in GRK2 expression levels

may indeed alter insulin signaling in vivo, we

studied whole-body insulin action and glucose

homeostasis in age-matched wild-type (Wt) and

GRK2+/-

male mice. These haploinsufficient mice

constitute a good model to recapitulate differences

in GRK2 expression in the same range to those

observed in pathological conditions, and also to

evaluate the potential of therapeutic strategies

aimed at decreasing GRK2 functionality in vivo.

Whereas GRK2 knock-out mice die in utero with

a marked cardiac hypoplasia (30), and the cardiac

phenotype of GRK2+/-

mice has been well

characterized (31;32), insulin signaling has not

been investigated so far in these animals.

Since TNFα infusion induces insulin

resistance in murine models in vivo (24;33),

we examined insulin sensitivity by GTTs and

ITTs in Wt and GRK2+/-

3-month-old male

mice after a 48h treatment with TNFα (Fig.

4A and 4C). In Wt, but not in GRK2+/-

mice,

TNFα produced a significant 29% increase in

fasting blood glucose levels (Table 1).

Moreover, the additional pronounced

hyperglycemic effect of TNFα after glucose

injection observed in Wt mice was attenuated

in GRK2+/-

mice (Fig. 4A). The improved

glucose tolerance observed was not due to an

increase in circulating insulin because

GRK2+/-

mice had lower circulating insulin

levels than Wt mice in both the fasted state

(Table 1) and during the GTT (Fig. 4B).

These results suggest that the more efficient

glucose clearance observed in GRK2+/-

mice

is caused by enhanced peripheral insulin

sensitivity. In line with this notion, the

hypoglycemic effect of insulin injection was

impaired upon TNFα treatment in Wt animals

but not in GRK2+/-

mice (Fig. 4C).

Wt mice treated with TNFα showed a

complete impairment in insulin-induced AKT

phosphorylation in skeletal muscle, adipose

tissue and liver (Fig. 4D and Suppl. Fig. S2

and Suppl. Table 1), that was not detected in

GRK2+/-

mice. Interestingly, TNFα

substantially increased GRK2 protein content

in eWAT and muscle of Wt mice (by 82%

and 42% respectively, Fig. 4D and Suppl. Fig.

S2 and Suppl. Table 1), whereas the amount

of GRK2 remained unaltered (in liver and

muscle) or below basal Wt levels (adipose)

upon TNFα treatment in GRK2+/-

mice (Fig.

4D, Suppl. Fig S2 and Suppl. Table 1).

Altogether, these results suggest that GRK2 is

a negative modulator of insulin sensitivity and

that lower GRK2 levels protect against the

systemic insulin resistance induced by TNFα.

GRK2 downregulation prevents the

development of aging-associated insulin

resistance. In the model of aging-associated


insulin resistance, GRK2 downregulation

caused no differences in the GTTs or ITTs of

young (3-month-old) male mice (Fig. 5A-C),

while in adult 9-month-old GRK2+/-

male

mice glucose tolerance was improved

compared to Wt (Fig. 5D). A similar tendency

had already been observed at 5 months of age

in GRK2+/-

mice (Suppl. Fig. S1). Aging also

induced a 57% increase in fasting glucose

concentration in Wt mice, limited to 29% in

GRK2+/-

mice (Table 1). Circulating levels of

insulin were higher in 9-month-old Wt than in

GRK2+/-

mice in response to an identical

glucose load (Fig. 5E). Consistently, GRK2+/-

mice exhibited an enhanced decline in blood

glucose levels upon administration of insulin

(Fig. 5F), and lower fasting serum insulin

levels (Table 1). Altogether these results

suggest increased whole-body insulin

sensitivity in GRK2+/-

mice, in agreement

with the values obtained for the HOMA index

(Table 1).

Insulin-stimulated AKT phosphorylation was

markedly impaired with age in eWAT, liver

and muscle in Wt mice, while GRK2+/-

animals maintained a robust activation of this

pathway (Fig. 5G and Suppl. Fig. S2 and

Suppl. Table 1). Interestingly, in adult

animals, GRK2 protein expression was

enhanced by 33% and 68% in eWAT and

muscle, respectively, compared to young mice

(Figs 3A, 5G and Suppl. Fig. S2 and Suppl.

Table 1), whilst GRK2 levels in adult

GRK2+/-

mice were below the expression

detected at 3 months of age in Wt animals in

all tissues examined (Fig. 5G and Suppl. Fig.

S2 and Suppl. Table 1). Overall, these data

suggest there is a threshold above which the

enhanced GRK2 expression observed with

age markedly affects insulin sensitivity in

vivo.

GRK2+/-

mice display protection against

high fat diet-induced insulin resistance and

obesity. After a HFD regime for 12 weeks,

Wt mice gained significantly more weight

than GRK2+/-

mice (Fig. 6A). The overall

increase in body weight was of 67% in Wt

and only 53% in GRK2+/-

mice (Fig. 6B),

without changes in food intake (Fig. 6C).

Consistently, even when adipocyte diameter

was increased in both groups, adiposity in

GRK2 hemizygous mice did not go beyond a

certain threshold that was exceeded only in

WT mice, with the size of HFD-fed GRK2+/-

epididymal adipocytes being 45% inferior

than that of Wt mice (Fig. 6D).

GTT analysis indicated a clear deterioration

in glucose metabolism in Wt but not in HFD-

fed GRK2+/-

mice. In the HFD-fed state,

GRK2+/-

mice did not develop glucose

intolerance (Fig. 6E), since we detect no

statistically significant changes in glucose

area between chow diet (CD)-fed and HFD-

fed GRK2+/-

mice, which showed lower blood

glucose levels (Table 1) and an enhanced

reduction in blood glucose induced by insulin

(Fig. 6F). HFD caused a marked increase in

circulating levels of glucose, insulin and TG

after 12 weeks of feeding (Table 1) in Wt

mice whereas these changes were much more

modest in GRK2+/-

animals. These parameters

were not altered with CD in either type of

animals (data not shown). In sum, HFD

promoted a marked insulin resistance state in

Wt mice, in accordance with the observed

increase in HOMA values (Table 1), whereas

insulin sensitivity was preserved in GRK2+/-

mice.

Insulin-induced AKT phosphorylation was

dramatically decreased in adipose tissue, liver

and muscle of Wt but not HFD-fed GRK2+/-

mice (Fig. 6G and Suppl. Fig. S2 and Suppl.

Table 1). On the other hand, in agreement

with data in Fig. 3A, GRK2 protein levels

were significantly increased in eWAT (84%),

liver (26%) and muscle (63%) of Wt but not

GRK2+/-

mice fed on a HFD (Fig. 6G and

Suppl. Fig. S2 and Suppl. Table 1). These

results indicate that even in different cellular

settings, whenever glucose intolerance

develops, GRK2+/-

mice always maintain the

levels of GRK2 protein below those detected


in Wt and thus below a given threshold that

helps to prevent the development of insulin

insensitivity

GRK2+/-

mice exhibit reduced adiposity . In

addition to the data shown in Fig. 6A-C, a

detailed weekly analysis of the weight of Wt

and GRK2+/-

animals from weaning to 15

months of age (Fig. 7A) revealed less weight

gain for both male and female GRK2+/-

mice

(Fig. 7B), without differences in daily food

intake (Fig. 7C). Body weight differences

were detected as early as at 5 months of age

(Fig. 7A), and at 9 months of age, when some

Wt mice are visibly obese (Fig. 7D), the

average body weight gain was 65% and 56%

in male and female Wt mice, respectively, but

only 56% and 43% in GRK2+/-

mice (Fig.

7B). Body fat distribution was studied by

Magnetic Nuclear Resonance Imaging

(MNRI) (Fig. 7E), showing that 9-month-old

GRK2+/-

mice had 33% less overall fat and

smaller fat depots than age-matched Wt

littermates (Fig. 7F). Epididymal fat mass was

reduced by 30% in GRK2+/-

compared to Wt

mice at 9 months of age (Fig. 7G), and

GRK2+/-

mice exhibited reduced adipocytic

size in epididymal (3-fold), retroperitoneal

(4.6-fold) and inguinal (3-fold) fat (Fig. 7H).

Interestingly, a decrease in circulating leptin

levels was also detected in GRK2+/-

animals at

9 months of age (Table 1). These results

indicate that downregulation of GRK2 is

sufficient to induce a lean phenotype, what

uncovers a previously unidentified role for

GRK2 in the regulation of adiposity.

DISCUSSION

In this report we uncover GRK2 as a key

modulator of insulin sensitivity in vivo. In

cultured adipocytes and myoblasts, increased

GRK2 or GRK2-K220R levels inhibit insulin-

stimulated glucose uptake and signaling in a

kinase-activity independent manner, by

mechanisms involving the formation of

dynamic GRK2/IRS1 complexes. GRK2

expression is enhanced by approximately 2-

fold in insulin-resistant human adipocytes, in

blood mononuclear cells from insulin-

resistant patients and in adipose and muscle

tissues in either TNFα, aging or HFD-induced

insulin resistance models. Importantly,

GRK2+/-

mice maintain glucose tolerance and

insulin signaling in the major insulin-

responsive tissues under such experimental

conditions, suggesting that enhanced GRK2

expression above a certain threshold markedly

impairs insulin sensitivity in vivo. Finally, we

find that GRK2 levels also regulate adiposity

during aging. Since GRK2 haploinsuficient

animals show a phenotype quite similar to

mice deficient in other well established

negative regulators of insulin signaling such

as PTP1B (24;33;34), GRK2 can be regarded

as a bona fide novel physiological regulator of

insulin action and leanness.

Previous reports have indicated that GRK2

could act as an inhibitor of insulin action in

cellular models. Binding of GRK2 to Gαq/11

impairs insulin-stimulated glucose uptake in

3T3L1 pre-adipocytes (19), and enhanced

GRK2 levels favour insulin resistance

induced by endothelin-1 in 3T3L1 cells (29)

or by chronic β-adrenergic receptor

stimulation in HEK-293 cells (28). GPCR

agonists promote GRK2 interaction with Gαq

and also with IRS1, resulting in decreased

insulin-stimulated glucose transport, IRS-

serine phosphorylation and IRS1 degradation

(29). Our data in cultured cells as well as in

adipose tissue in vivo show that IRS1 levels

as well as the amount of basal GRK2-IRS1

complexes depend on GRK2 expression and

that insulin stimulation rapidly disrupts basal

IRS1/GRK2 complexes, what is hampered in

the presence of elevated levels of GRK2.

Overall, these data suggest that altered GRK2

levels could lead to modulation of insulin

signals through GRK2-Gαq/11 binding,

GRK2-IRS1 association, and altered

endothelin-1 or β-adrenergic-mediated

transmodulation of the insulin pathway. The

precise contribution of such mechanisms in


different physiological conditions remains to

be investigated.

Interestingly, β-arrestin2, another member of

the GPCR signal transduction pathway,

positively regulates insulin sensitivity by

serving as a scaffold of AKT and Src to the

IR (35). β-arrestin2 levels are downregulated

in liver and muscle in animal models and

patients with insulin resistance. Therefore,

simultaneous up-regulation of GRK2 and

down-regulation of β-arrestin2 in insulin-

resistant conditions could lead to major

alterations in the insulin signaling pathways

and in GPCR-IR cross-talk. It is worth noting

that elevated levels of insulin and IGF-1 have

been shown to exert differential effects on

GRK2 and β-arrestin2 expression (36;37).

Enhanced GRK2 levels could alter the

proposed novel IR-arrestin signaling pathway

by re-directing arrestin to other interactors,

such as GPCR or, given the reported direct

interactions of the kinase with AKT or Src, by

inhibiting the formation of the

arrestin/AKT/Src complexes.

GRK2 is degraded by the proteasome

pathway by associating with the Mdm2 E3-

ubiquitin ligase, and activation of the

PI3K/AKT pathway by IGF-1 blocks Mdm2-

mediated GRK2 degradation, leading to

enhanced GRK2 levels (38). Consistently,

chronic insulin has been recently shown to

increase GRK2 levels in HEK-293 and liver

FL83B cells (20;28) what is in agreement

with the upregulation of GRK2 we observe

after insulin treatment in human adipocytic

cells. Thus, it is tempting to suggest that the

hyperinsulinemia associated to aging, HFD-

or TNFα-induced insulin resistant conditions,

and clinically to obesity and type-2-diabetes,

would trigger the observed up-regulation of

GRK2 in such conditions. Consistent with our

findings, enhanced GRK2 expression has

been reported in tissues from obese Zucker

rats, a model of insulin resistance and

hyperinsulinemia (39).

An altered cytokine expression pattern typical

of insulin-resistant states could also contribute

to enhance GRK2 expression levels. TNFα or

IL-6 increase the expression of other negative

modulators of the insulin signalling cascade

like PTP1B (24;40), and we find that TNFα

also enhances GRK2 protein content in

adipose tissue and muscle of Wt mice in the

presence of mild hyperinsulinemia. As in

GRK2+/-

mice, PTP1B-deficient animals also

exhibit protection against insulin resistance

induced by TNFα (24), or IL-6 (40). Pro-

inflammatory mediators can modulate

endogenous GRK2 expression in a cell-type

specific fashion (41), and an increase in

GRK2 protein is found upon chronic IL-1β

treatment (26;42). Interestingly, GRK2

appears to have a relevant role in the

aetiology and/or in the development of

several inflammatory diseases such as

multiple sclerosis, and autoimmune arthritis

(43). A role for GRK2 in states of insulin

resistance associated to inflammatory diseases

deserves further investigation.

The fact that the 50% down-regulation of

GRK2 levels is sufficient per se to protect

against TNFα, aging or HFD-induced

alterations in glucose homeostasis and insulin

signaling strongly supports that this kinase is

a key modulator of insulin sensitivity in vivo

and suggests new therapeutic strategies

against type-2-diabetes and obesity. In fact, in

animal models of type-2-diabetes, GRK2

inhibition through systemic delivery of small

peptides derived from its catalytic domain

results in improved glucose homeostasis

(28;44). It remains to be established whether

inhibition of catalytic activity,

downregulation of protein expression or

targeted disruption of the specific interaction

of GRK2 with insulin signaling pathway

components are the more appropriate

strategies for specifically improving insulin

sensitivity.

The GRK2+/-

mice constitute a good model

that recapitulates a sustained systemic


inhibition of kinase functionality. We report

here that GRK2 levels are important

modulators of age- and diet-induced

adiposity. GRK2+/-

mice gained less weight,

showed diminished adipocyte size with a

HFD, and adult hemizygous animals

displayed reduced adiposity and lower

circulating levels of insulin and leptin. Aging

is associated with fat mass accretion and with

decreased peripheral insulin sensitivity in

humans and rodents (45), and impaired

insulin actions in adipose tissue could

represent a key step leading to the overall

insulin resistance characteristic of adult

and/or obese animals (46). Adipocyte-

conditioned medium impairs insulin signaling

in muscle cells (22) and hepatocytes (47), and

mice that exhibit reduced adiposity typically

display improved glucose tolerance and

increased insulin sensitivity (46). Therefore, it

is possible that part of the insulin

hypersensitivity detected in GRK2+/-

mice and

the protection afforded to the development of

insulin resistant conditions in these animals

could be related to the observed changes in

adiposity. Future experiments involving

tissue-specific downregulation will provide

new insight into the understanding of the role

of GRK2 in obesity and insulin resistance.

Author contributions: L.G-G & R. V-V.,

researched data, contributed to discussion;

I.N-V., wrote manuscript, researched data,

contributed to discussion; M.J-P., researched

data; G.Z. & J.D., collected samples,

reviewed/edited manuscript. C.M., F.M. Jr.,

M.L. & S. F-V., contributed to discussion,

wrote/edited manuscript.

ACKNOWLEDGMENTS

This work is dedicated to the memory of Prof.

Margarita Lorenzo, who passed away April 7,

2010, at the age of 51.

This work was supported by Grants

BFU2008-04043 (to M.L), SAF2008-0552 (to

F.M) and SAF2007-62553 (to G.Z.) from

Ministerio de Ciencia e Innovación, Spain, S-

SAL-0159-2006 from Comunidad de Madrid,

Spain (to M.L. and F.M), Fundación Ramón

Areces (to F.M), the European Union

(InGenious HyperCare, grant LSHM-CT-

2006-037093 (to J.D., through the agreement

between the Foundation for Applied Medical

Research (FIMA) and “UTE project CIMA”),

The Cardiovascular Network (RECAVA,

RD06-0014/0037 and 0014/008 to F.M and

J.D., respectively), CIBER de Diabetes y

Enfermedades Metabólicas Asociadas, and

PS09/01208 (to C.M.) from Ministerio

Sanidad y Consumo-Instituto Carlos III,

Spain. We also acknowledge the support of

COST Action BM0602 from the European

Commission (to M.L.). The authors thank to

Dr. Oscar Beloqui from the University Clinic

of Navarra, for recollection of clinical data of

studied patients.

REFERENCES

1. Biddinger SB, Kahn CR. From mice to men. insights into the insulin resistance syndromes.

Annu Rev Physiol 2006;68:123-158

2. White MF. Insulin signaling in health and disease. Science 2003;302:1710-1711

3. Hoehn KL, Hohnen-Behrens C, Cederberg A, Wu LE, Turner N, Yuasa T, Ebina Y, James

DE. IRS1-independent defects define major nodes of insulin resistance. Cell Metab

2008;7:421-433

4. Trayhurn P, Wood IS. Signalling role of adipose tissue: adipokines and inflammation in

obesity. Biochem Soc Trans 2005;33:1078-1081

5. Waki H, Tontonoz P. Endocrine functions of adipose tissue. Annu Rev Pathol 2007;2:31-

56


6. de Luca C, Olefsky JM. Inflammation and insulin resistance. FEBS Lett 2008;582:97-105

7. Hernandez R, Teruel T, de Alvaro C, Lorenzo M. Rosiglitazone ameliorates insulin

resistance in brown adipocytes of Wistar rats by impairing TNF-alpha induction of p38 and

p42/p44 mitogen-activated protein kinases. Diabetologia 2004; 47:1615-1624

8. Fernandez-Veledo S, Nieto-Vazquez I, Vila-Bedmar R, Garcia-Guerra L, Alonso-

Chamorro M, Lorenzo M. Molecular mechanisms involved in obesity-associated insulin

resistance: therapeutical approach. Arch Physiol Biochem 2009;115:227-239

9. de Alvaro C, Teruel T, Hernandez R, Lorenzo M. Tumor necrosis factor alpha produces

insulin resistance in skeletal muscle by activation of inhibitor kappaB kinase in a p38

MAPK-dependent manner. J Biol Chem 2004;279:17070-17078

10. Nieto-Vazquez I, Fernandez-Veledo S, Kramer DK, Vila-Bedmar R, Garcia-Guerra L,

Lorenzo M. Insulin resistance associated to obesity: the link TNF-alpha. Arch Physiol

Biochem 2008;114:183-194

11. Fernandez-Veledo S, Vila-Bedmar R, Nieto-Vazquez I, Lorenzo M. c-Jun N-terminal

kinase 1/2 activation by tumor necrosis factor-alpha induces insulin resistance in human

visceral but not subcutaneous adipocytes: reversal by liver X receptor agonists. J Clin

Endocrinol Metab 2009;94:3583-3593

12. Huang S, Czech MP. The GLUT4 glucose transporter. Cell Metab 2007;5:237-252

13. Zaid H, Antonescu CN, Randhawa VK, Klip A. Insulin action on glucose transporters

through molecular switches, tracks and tethers. Biochem J 2008;413:201-215

14. Kanzaki M, Watson RT, Artemyev NO, Pessin JE. The trimeric GTP-binding protein

(G(q)/G(11)) alpha subunit is required for insulin-stimulated GLUT4 translocation in

3T3L1 adipocytes. J Biol Chem 2000;275:7167-7175

15. Imamura T, Vollenweider P, Egawa K, Clodi M, Ishibashi K, Nakashima N, Ugi S, Adams

JW, Brown JH, Olefsky JM. G alpha-q/11 protein plays a key role in insulin-induced

glucose transport in 3T3-L1 adipocytes. Mol Cell Biol 1999;19:6765-6774

16. Usui I, Imamura T, Huang J, Satoh H, Olefsky JM. Cdc42 is a Rho GTPase family member

that can mediate insulin signaling to glucose transport in 3T3-L1 adipocytes. J Biol Chem

2003;278:13765-13774

17. Jurado-Pueyo M, Campos PM, Mayor F, Murga C. GRK2-dependent desensitization

downstream of G proteins. J Recept Signal Transduct Res 2008;28:59-70

18. Ribas C, Penela P, Murga C, Salcedo A, Garcia-Hoz C, Jurado-Pueyo M, Aymerich I,

Mayor F, Jr.. The G protein-coupled receptor kinase (GRK) interactome: role of GRKs in

GPCR regulation and signaling. Biochim Biophys Acta 2007;1768:913-922

19. Usui I, Imamura T, Satoh H, Huang J, Babendure JL, Hupfeld CJ, Olefsky JM. GRK2 is an

endogenous protein inhibitor of the insulin signaling pathway for glucose transport

stimulation. EMBO J 2004;23:2821-2829

20. Shahid G, Hussain T. GRK2 negatively regulates glycogen synthesis in mouse liver FL83B

cells. J Biol Chem 2007;282:20612-20620

21. Wabitsch M, Bruderlein S, Melzner I, Braun M, Mechtersheimer G, Moller P. LiSa-2, a

novel human liposarcoma cell line with a high capacity for terminal adipose differentiation.

Int J Cancer 2000;88:889-894

22. Fernandez-Veledo S, Nieto-Vazquez I, de Castro J, Ramos MP, Bruderlein S, Moller P,

Lorenzo M. Hyperinsulinemia induces insulin resistance on glucose and lipid metabolism

in a human adipocytic cell line: paracrine interaction with myocytes. J Clin Endocrinol

Metab 2008;93:2866-2876


23. Fortuno A, Olivan S, Beloqui O, San JG, Moreno MU, Diez J, Zalba G. Association of

increased phagocytic NADPH oxidase-dependent superoxide production with diminished

nitric oxide generation in essential hypertension. J Hypertens 2004;22:2169-2175

24. Nieto-Vazquez I, Fernandez-Veledo S, de Alvaro C, Rondinone CM, Valverde AM,

Lorenzo M. Protein-Tyrosine Phosphatase 1B-Deficient Myocytes Show Increased Insulin

Sensitivity and Protection Against Tumor Necrosis Factor-{alpha}-Induced Insulin

Resistance. Diabetes 2007;56:404-413

25. Jimenez-Sainz MC, Murga C, Kavelaars A, Jurado-Pueyo M, Krakstad BF, Heijnen CJ,

Mayor F, Jr., Aragay AM. G protein-coupled receptor kinase 2 negatively regulates

chemokine signaling at a level downstream from G protein subunits. Mol Biol Cell

2006;17:25-31

26. Kleibeuker W, Jurado-Pueyo M, Murga C, Eijkelkamp N, Mayor F, Jr., Heijnen CJ,

Kavelaars A. Physiological changes in GRK2 regulate CCL2-induced signaling to ERK1/2

and Akt but not to MEK1/2 and calcium. J Neurochem 2008; 104:979-992

27. Iaccarino G, Barbato E, Cipolletta E, de Amicis V, Margulies KB, Leosco D, Trimarco B,

Koch WJ. Elevated myocardial and lymphocyte GRK2 expression and activity in human

heart failure. Eur Heart J 2005;26:1752-1758

28. Cipolletta E, Campanile A, Santulli G, Sanzari E, Leosco D, Campiglia P, Trimarco B,

Iaccarino G. The G protein coupled receptor kinase 2 plays an essential role in beta-

adrenergic receptor-induced insulin resistance. Cardiovasc Res 2009

29. Usui I, Imamura T, Babendure JL, Satoh H, Lu JC, Hupfeld CJ, Olefsky JM. G protein-

coupled receptor kinase 2 mediates endothelin-1-induced insulin resistance via the

inhibition of both Galphaq/11 and insulin receptor substrate-1 pathways in 3T3-L1

adipocytes. Mol Endocrinol 2005;19:2760-2768

30. Jaber M, Koch WJ, Rockman H, Smith B, Bond RA, Sulik KK, Ross J, Jr., Lefkowitz RJ,

Caron MG, Giros B. Essential role of beta-adrenergic receptor kinase 1 in cardiac

development and function. Proc Natl Acad Sci U S A 1996; 93:12974-12979

31. Penela P, Murga C, Ribas C, Tutor AS, Peregrin S, Mayor F, Jr.. Mechanisms of regulation

of G protein-coupled receptor kinases (GRKs) and cardiovascular disease. Cardiovasc Res

2006;69:46-56

32. Dorn GW. GRK mythology: G-protein receptor kinases in cardiovascular disease. J Mol

Med 2009;87:455-463

33. Elchebly M, Payette P, Michaliszyn E, Cromlish W, Collins S, Loy AL, Normandin D,

Cheng A, Himms-Hagen J, Chan CC, Ramachandran C, Gresser MJ, Tremblay ML,

Kennedy BP. Increased insulin sensitivity and obesity resistance in mice lacking the protein

tyrosine phosphatase-1B gene. Science 1999; 283:1544-1548

34. Klaman LD, Boss O, Peroni OD, Kim JK, Martino JL, Zabolotny JM, Moghal N, Lubkin

M, Kim YB, Sharpe AH, Stricker-Krongrad A, Shulman GI, Neel BG, Kahn BB. Increased

energy expenditure, decreased adiposity, and tissue-specific insulin sensitivity in protein-

tyrosine phosphatase 1B-deficient mice. Mol Cell Biol 2000;20:5479-5489

35. Luan B, Zhao J, Wu H, Duan B, Shu G, Wang X, Li D, Jia W, Kang J, Pei G. Deficiency of

a beta-arrestin-2 signal complex contributes to insulin resistance. Nature 2009;457:1146-

1149

36. Dalle S, Imamura T, Rose DW, Worrall DS, Ugi S, Hupfeld CJ, Olefsky JM. Insulin

induces heterologous desensitization of G-protein-coupled receptor and insulin-like growth

factor I signaling by downregulating beta-arrestin-1. Mol Cell Biol 2002;22:6272-6285


37. Hupfeld CJ, Dalle S, Olefsky JM. Beta -Arrestin 1 down-regulation after insulin treatment

is associated with supersensitization of beta 2 adrenergic receptor Galpha s signaling in

3T3-L1 adipocytes. Proc Natl Acad Sci U S A 2003;100:161-166

38. Salcedo A, Mayor F, Jr., Penela P. Mdm2 is involved in the ubiquitination and degradation

of G-protein-coupled receptor kinase 2. EMBO J 2006;25:4752-4762

39. Trivedi M, Lokhandwala MF. Rosiglitazone restores renal D1A receptor-Gs protein

coupling by reducing receptor hyperphosphorylation in obese rats. Am J Physiol Renal

Physiol 2005;289:F298-F304

40. Nieto-Vazquez I, Fernandez-Veledo S, de Alvaro C, Lorenzo M. Dual role of interleukin-6

in regulating insulin sensitivity in murine skeletal muscle. Diabetes 2008; 57:3211-3221

41. Ramos-Ruiz R, Penela P, Penn RB, Mayor F, Jr.. Analysis of the human G protein-coupled

receptor kinase 2 (GRK2) gene promoter: regulation by signal transduction systems in

aortic smooth muscle cells. Circulation 2000;101:2083-2089

42. Mak JC, Hisada T, Salmon M, Barnes PJ, Chung KF. Glucocorticoids reverse IL-1beta-

induced impairment of beta-adrenoceptor-mediated relaxation and up-regulation of G-

protein-coupled receptor kinases. Br J Pharmacol 2002;135:987-996

43. Vroon A, Heijnen CJ, Kavelaars A. GRKs and arrestins: regulators of migration and

inflammation. J Leukoc Biol 2006;80:1214-1221

44. Anis Y, Leshem O, Reuveni H, Wexler I, Ben SR, Yahalom B, Laster M, Raz I, Ben SS,

Shafrir E, Ziv E. Antidiabetic effect of novel modulating peptides of G-protein-coupled

kinase in experimental models of diabetes. Diabetologia 2004;47:1232-1244

45. Escriva F, Gavete ML, Fermin Y, Perez C, Gallardo N, Alvarez C, Andres A, Ros M,

Carrascosa JM. Effect of age and moderate food restriction on insulin sensitivity in Wistar

rats. role of adiposity. J Endocrinol 2007;194:131-141

46. Sabio G, Das M, Mora A, Zhang Z, Jun JY, Ko HJ, Barrett T, Kim JK, Davis RJ. A stress

signaling pathway in adipose tissue regulates hepatic insulin resistance. Science

2008;322:1539-1543

47. Wang Z, Lv J, Zhang R, Zhu Y, Zhu D, Sun Y, Zhu J, Han X. Co-culture with fat cells

induces cellular insulin resistance in primary hepatocytes. Biochem Biophys Res Commun

2006;345:976-983


Table 1. Metabolic parameters of Wt and GRK2+/-

male mice.

Serum from Wt and GRK2+/-

3 and 9-month-old male mice, fed on a HFD for 12 weeks or adult male mice treated with TNFα as in

Fig. 4 was collected, and the indicated metabolic parameters were measured as detailed in Methods. The HOMA index was calculated

as the product of the fasting plasma insulin level (mU/mL) and the fasting plasma glucose level (mmol/L), divided by 22.5. Values are

expressed as the mean±SEM of measurements obtained for 8-18 animals for each group. Differences between Wt vs GRK2+/-

mice are

expressed by *, and differences between 3-month-old mice vs 9-month-old mice, fed a HFD or treated with TNFα are expressed by §.

*, § p<0.05.

3 months 9 months HFD TNFα

Parameter

Wt GRK2+/- Wt GRK2+/- Wt GRK2+/- Wt GRK2+/-

Blood glucose (mg/dl)

Fed 155,4 ± 5,9 152 ± 6,3 170,2 ± 6,7 § 152,7 ± 6,4 * 184,4 ± 9,7 § 168 ± 5,7 *§ 148 ± 6,5 135 ± 7,1

Fasted 81,6 ± 3,6 81,6 ± 2,6 128,3 ± 6,0 § 105,6 ± 8,3 *§ 110 ± 3,7 § 89,6 ± 4,0 *§ 105,8±6,2 § 83 ± 2,3 *

Serum Insulin (ng/ml)

Fed 0,65 ± 0,08 0,49 ± 0,09 0,98 ± 0,05 § 0,60 ± 0,02 * 2,50 ± 0,30 § 1,03 ± 0,01 *§ - -

Fasted 0,13 ± 0,02 0,13 ± 0,01 0,61 ± 0,05 § 0,44 ± 0,04 *§ 0,54 ± 0,02 § 0,16 ± 0,01 *§ 0,26 ± 0,02 § 0,15 ± 0,02 *

Triglycerides (µg/µl)

Fed 0,046 ± 0,002 0,054 ± 0,001* 0,038 ± 0,005 § 0,047 ± 0,006 § 0,140 ± 0,020 § 0,064 ± 0,001 *§ - -

Fasted 0,018 ± 0,002 0,011 ± 0,001* 0,035 ± 0,001 § 0,033 ± 0,002 § 0,060 ± 0,002 § 0,045 ± 0,007 *§ - -

Leptin(ng/ml)

Fed 6,7 ± 1,5 6,6 ± 1,3 8,4 ± 1,7 6,1 ± 1,7 14 ± 3,6 § 13,1 ± 3,0 § - -

HOMA 0,63 ± 0,07 0,62 ± 0,08 5,02 ± 0,63 § 2,53 ± 0,16 *§ 3,52 ± 0,42 § 0,85 ± 0,07 * 1,63 ± 0,09 § 0,74 ± 0,06 *

* Wt vs GRK2+/-; P<0.05

§ 3 months vs 9 months, HFD, TNFαααα; P<0.05


FIGURE LEGENDS

Fig. 1. Modulation on insulin sensitivity by GRK2

Immortalized murine white adipocytes and C2C12 myocytes were transiently transfected with

empty vector (control, C), pcDNA3-GRK2-neo (+), the catalytically inactive pcDNA3-GRK2-

K220R (K) or shGRK2-RNAi (-), to upregulate or knock-down GRK2 expression, respectively.

(A) Lysates were analyzed by Western-blot with the corresponding antibodies against GRK2 and

β-actin. For examples of full-length blots see Suppl. Fig. S3. (B) Adipocytes and myocytes were

cultured for 24h in serum-free and low glucose medium and stimulated in absence or presence of

10 nM insulin (Ins) for 30 min. Glucose uptake was measured during the last 10 min by

incorporation of 2-deoxy-glucose into the cells. Results were expressed as percentage of

stimulation over basal of control cells (adipocytes: 5.3±0.3 pmol glucose/mg protein/10 min;

myocytes: 45.8±3.2 pmol glucose/mg protein/10 min) and are means ± SEM of 5 independent

experiments. (C) Adipocytic and (D) myocytic cell lines were serum-starved overnight and then

incubated in absence or presence of 10 nM insulin for 10 min. 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 β-actin. Representative

immunoblots of 4-6 independent experiments and densitometric analysis are shown in A, C and

D. *, p < 0.01. For examples of full-length blots see Suppl. Fig. S3. Data in A, C and D were

normalized with the indicated controls and results are expressed as % over basal (control cells

transfected with empty vector).

Fig. 2. Modulation of insulin signalling by GRK2 levels involves the formation of dynamic

GRK2/IRS1 complexes.

Immortalized murine white adipocytes were transiently transfected with empty vector (control,

C), pcDNA3-GRK2-neo (+), or shGRK2-RNAi (-), to upregulate or knock-down GRK2

expression, respectively. (A) Adipocytic cell lines were serum-starved overnight and then

incubated in absence or presence of 10 nM insulin for 10 min. Total protein from adipocytic cells

(1mg) was immunoprecipitated with the anti-IRS1 or anti-IgG antibodies, and the resulting

immune complexes analyzed by Western-blot with the corresponding antibodies against GRK2

and IRS1. (B) Samples of eWAT were obtained at the indicated times from Wt and GRK2+/-

adult male mice undergoing Ins infusion (1 IU/kg body weight) as detailed in Methods. Lysates

were immunoprecipitated with an anti-IRS1 antibody, and the resulting immune complexes

analyzed by Western-blot with the corresponding antibodies against GRK2 and IRS1. (C) The

same lysates were analyzed by Western-blot with the corresponding antibodies against total

and/or phosphorylated forms of IRS1(Tyr 608), AKT(Ser473) and β-actin. Representative

immunoblots of 4-6 independent experiments and densitometric analysis are shown. Data were

normalized with the indicated controls, and results obtained in non-stimulated control cells (A)

or Wt animals (B, C) taken as 100%. *, p < 0.01.

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 TNFα (0.1 µg/kg body weight) for 48h; (B) eWAT from wild-type (Wt) and ob/ob

mice; (C) insulin sensitive and insulin resistant human adipocytes, and (D) PBMCs collected

from blood samples of insulin-sensitive individuals and insulin-resistant patients (see Suppl.

Table 2) were lysed and subjected to immunoblot analysis with antibodies against GRK2 and β-

actin as a loading control. Data in A, B and C are mean±SEM of 4 independent experiments.


Data in A were normalized with the indicated control and results expressed as % over basal (3

month-Wt mice). Results in D represent the mean±SEM of 10 controls and 25 metabolic

syndrome patients. Representative blots are shown. * p < 0.01.

Fig. 4. GRK2+/-

mice do not develop TNFα-induced insulin resistance

Wt (white symbols, solid lines) and GRK2+/-

(black symbols, dotted lines) adult male mice were

treated with TNFα (0.1 µg/kg body weight, circles) or vehicle (100 µl PBS plus 0.1% BSA,

squares) for 48 h. (A) GTTs were performed on animals that had been fasted for 24h and were

given an injection of glucose (2 g/kg body weight). (B) Circulating insulin levels during the

GTTs. (C) ITTs were performed on fed male mice that had received insulin (0.8 IU/kg body

weight). Glucose concentration was determined in blood samples obtained from the tail vein.

Results are means±SEM of 8 animals for each group. Histograms on the right show the

incremental area under the curve. *, p < 0.01. (D) Wild-type (Wt) and GRK2+/-

male mice were

treated or not with insulin (1 IU/kg body weight) for 15 min. and eWAT, liver and muscle were

removed. Lysates were subjected to Western-blot with the indicated antibodies against total and

phosphorylated AKT(Ser473), GRK2 and β-actin. Representative immunoblots out of 4

independent experiments are shown. See Suppl. Fig. S2 and Suppl. Table 1 for detailed

quantification of these data. For examples of full-length blots see Suppl. Fig. S4.

Fig. 5. GRK2 downregulation prevents the development of aging-associated insulin

resistance.

(A) GTTs were performed on Wt (white circles, solid lines) and GRK2+/-

(black circles, dotted

lines) 3-month-old male mice as in Fig. 4A. (B) Circulating insulin levels during the GTTs. (C)

ITTs were performed on Wt (white circles) and GRK2+/-

(black circles) 3-month-old male mice

as in Fig. 4C. (D, E, F) GTTs and ITTs were performed as in previous panels in Wt (white

circles) and GRK2+/-

(black circles) 9-month-old male mice. *, p < 0.01. (G) Lysates from Wild-

type (Wt) and GRK2+/-

3 and 9-month-old male mice treated or not with insulin, were processed

as in Fig. 4D. Representative immunoblots out of 4 independent experiments are shown. See

Suppl. Fig. S2 and Suppl. Table 1 for detailed quantification of these data.

Fig. 6. GRK2+/-

mice display protection against diet-induced obesity. Wt (white symbols, solid lines) and GRK2

+/- (black symbols, dotted lines) male mice were fed a

CD or HFD for 12 weeks. (A) Body weight evolution and (A) Body weight evolution and (B)

percentage of change in body weight during the 12 weeks of treatment. (C) Daily food intake on

CD or HFD. (D) Paraffin-embedded sections of epididymal fat pads from Wt and GRK2+/-

male

mice fed on CD or HFD stained with haematoxylin and eosin (Magnification 10x; scale bar

50µm for all pictures). Relative adipocyte size was calculated as detailed in Methods. Data are

mean±SEM of 10-20 animals per group. (E) GTTs (circles, HFD; squares, CD) and (F) ITTs

were performed as in Fig. 4A and 4C, respectively. *, p < 0.01. (G) Lysates from Wt and

GRK2+/-

male mice fed CD or HFD for 12 weeks treated or not with insulin were processed as in

Fig. 4D. Representative immunoblots out of 4 independent experiments are shown. See Suppl.

Fig. S2 and Suppl. Table 1 for detailed quantification of these data.

Fig. 7. GRK2+/-

mice exhibit reduced adiposity.

(A) Body weight evolution in male or female Wt (white circles, solid lines) and GRK2+/-

(black

circles, dotted lines) mice between 2 and 15 months of age. (B) Percentage of change in body


weight during 13 months. (C) Daily food intake in 3 and 9-month-old male Wt and GRK2+/-

mice. (D) Representative photograph showing 9-month-old Wt or GRK2+/-

male mice (E)

Adiposity in 9 month-old male mice measured by MNRI; white represents areas with more than

50% fat. (F) Quantification of several MNRI measurements. Results were expressed as

proportion between fat volumes vs total mouse volume (fat vol/total vol). (G) eWAT weight in 3

and 9-month-old male Wt and GRK2+/-

mice. (H) Paraffin-embedded sections of WAT:

retroperitoneal (Ret), epididymal (Epi) and inguinal (Ing) fat pads from 9-month-old male Wt

and GRK2+/-

mice stained with haematoxylin and eosin. (Magnification 10x; scale bar 50µm for

all pictures). Relative adipocyte size was calculated as in Fig. 6D. Data are mean ± SEM of 5-30

animals per group. *, p < 0.01.

g protein-coupled receptor kinase 2...

Documents