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The FASEB Journal Research Communication

AMP-activated protein kinase mediates glucocorticoid-induced metabolic changes: a novel mechanism inCushings syndrome

Mirjam Christ-Crain,*,1 Blerina Kola,*,1 Francesca Lolli,* Csaba Fekete,

Dalma Seboek, Gabor Wittmann, Daniel Feltrin,* Susana C. Igreja,* Sharon Ajodha,*Judith Harvey-White, George Kunos, Beat Muller, Francois Pralong, Gregory Aubert,

Giorgio Arnaldi, Gilberta Giacchetti, Marco Boscaro, Ashley B. Grossman,* andMarta Korbonits*,2

*Department of Endocrinology, William Harvey Research Institute, Barts and the London, QueenMarys School of Medicine, University of London, London, UK; Department of EndocrineNeurobiology, Institute of Experimental Medicine, Hungarian Academy of Sciences, Budapest,Hungary, and Tupper Research Institute and Department of Medicine, Division of Endocrinology,Diabetes, Metabolism and Molecular Medicine, Tufts-New England Medical Center, Boston,Massachusetts, USA; Division of Endocrinology, Diabetes and Clinical Nutrition, University Hospital,Basel, Switzerland; Laboratory of Physiologic Studies, National Institute on Alcohol Abuse and

Alcoholism, U.S. National Institutes of Health, Bethesda, Maryland, USA;

Service dEndocrinologie,Diabetologie et Metabolism, Department of Internal Medicine, Centre Hospitalier UniversitaireVaudois, Lausanne, Switzerland; and Department of Internal Medicine, University of Ancona,Ancona, Italy

ABSTRACT Chronic exposure to glucocorticoid hor-mones, resulting from either drug treatment or Cush-ings syndrome, results in insulin resistance, centralobesity, and symptoms similar to the metabolic syn-drome. We hypothesized that the major metaboliceffects of corticosteroids are mediated by changes inthe key metabolic enzyme adenosine monophosphate-

activated protein kinase (AMPK) activity. Activation ofAMPK is known to stimulate appetite in the hypothala-mus and stimulate catabolic processes in the periphery.We assessed AMPK activity and the expression ofseveral metabolic enzymes in the hypothalamus, liver,adipose tissue, and heart of a rat glucocorticoid-excessmodel as well as inin vitrostudies using primary humanadipose and primary rat hypothalamic cell cultures, anda human hepatoma cell line treated with dexametha-sone and metformin. Glucocorticoid treatment inhib-ited AMPK activity in rat adipose tissue and heart, whilestimulating it in the liver and hypothalamus. Similardata were observedin vitroin the primary adipose and

hypothalamic cells and in the liver cell line. Metformin,a known AMPK regulator, prevented the corticosteroid-induced effects on AMPK in human adipocytes and rathypothalamic neurons. Our data suggest that glucocor-ticoid-induced changes in AMPK constitute a novelmechanism that could explain the increase in appetite,the deposition of lipids in visceral adipose and hepatictissue, as well as the cardiac changes that are allcharacteristic of glucocorticoid excess. Our data sug-gest that metformin treatment could be effective inpreventing the metabolic complications of chronic glu-cocorticoid excess.Christ-Crain, M., Kola, B., Lolli,

F., Fekete, C., Seboek, D., Wittmann, G., Feltrin, D.,Igreja, S. C., Ajodha, S., Harvey-White, J., Kunos, G.,Muller, B., Pralong, F., Aubert, G., Arnaldi, G., Giac-chetti, G., Boscaro, M., Grossman, A. B., Korbonits, M.AMP-activated protein kinase mediates glucocorticoid-induced metabolic changes: a novel mechanism inCushings syndrome. FASEB J. 22, 16721683 (2008)

Key Words: obesity insulin resistance lipid metabolism

Cushings syndrome results fromchronic exposureto excess glucocorticoid hormones, either produced bythe adrenal cortex or given exogenously. The chronichigh levels of glucocorticoid induce central obesity,systemic arterial hypertension, impaired glucose toler-ance, fatty liver, and dyslipidemia (in addition to ad-verse effects on the gastric mucosa, bone and mood),ultimately leading to a clinical condition which, to acertain extent, resembles the metabolic syndrome (1).These manifestations contribute to the increased car-

diovascular risk observed in patients with Cushingssyndrome, and to reduced life expectancy and qualityof life. Symptoms may persist even when the excessglucocorticoid exposure is normalized (2, 3). Obesityin Cushings syndrome is characterized by increased

1 These authors contributed equally to this work.2 Correspondence: Department of Endocrinology, John

Vane Science Centre, Barts and the London, Queen MarysSchool of Medicine, London EC1M 6BQ, UK. E-mail:[email protected]

doi: 10.1096/fj.07-094144

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and streptomycin (Sigma). All cell treatments were per-formed with dexamethasone 1 M diluted in ethanol, asdescribed above.

Primary hypothalamic rat cultures

Primary hypothalamic neuronal cell cultures were performedas described (21). Briefly, hypothalami were obtained from18-day-old rat fetuses, and cells were dispersed mechanically,plated at a density of 500 live cells/mm2 in 6-well plates

coated with 5 g/ml poly-d-lysine (Sigma), and grown inNeurobasalTM medium with 0.04% B27 supplement (LifeTechnologies, Inc.) containing 500 M glutamine and 25 Mglutamate (Sigma). Forty-eight hours after plating, 2 M araC(cytosine -d-arabinofuranoside; Sigma) was added to pre-

vent proliferation of non-neuronal cells. Half of the mediumwas then changed every fourth day, and all experiments wereperformed in 3- to 4-week-old culture. Experiments wereperformed after 6 h of starvation of the cells in DMEM (LifeTechnologies, Inc.) at 5.5 mM glucose with no FCS. Cells

were treated for 6 24 h with 1 M dexamethasone and 1 mMmetformin (Sigma).

Measurement of AMPK activity

AMPK activity assay has been previously described (13, 22).Proteins were extracted using appropriate lysis buffer (13,22), and protein content was determined using bicinchoninicacid assay (Pierce Biotechnology, Rockford, IL, USA). AMPK

was immunoprecipitated using 1 and 2 AMPK antibodies,and activity was measured using 32P incorporation into the

AMPK substrate SAMS (amino acid sequence: HMRSAMS-GLHLVKRR). Samples were assayed in duplicate, and eachsample also was assayed without the addition of the substrateSAMS as a negative control.

Reverse transcriptase-polymerase chain reaction (RT-PCR)

Total RNA from adipose tissues (100 mg) was extracted withQIAzol reagent (Qiagen, Crawley, UK) and from liver andheart tissues (30 mg) using the Promega SV isolation kit(Promega). cDNA was synthesized from 1 g RNA in a total

volume of 25 l using random primer (Roche, Burgess Hill,UK) and reverse transcriptase (Invitrogen). Levels of geneexpression were quantified using real-time PCR (ABI PRISM7900) with the human- and rat-specific assay-by-design primerand probe sets by Applied Biosystems (ABI, Warrington, UK)for rat-specific primers for sterol regulatory element-bindingprotein-1 (SREBP1c), fatty acid synthase (FAS), phosphoenol-pyruvate carboxykinase (PEPCK)-1c, G6P, hormone sensitivelipase (HSL), AMPK1, and AMPK2 (assay codes availableon request). Control reactions for RT (containing RNA butno RT enzyme) and PCR (containing PCR mixture but no

cDNA) were run together with samples. All gene expressionassays have FAM TM reporter dye at the 5 end of theTaqMan MGB probe and a nonfluorescent quencher at the3 end of the probe. The TaqMan MGB probes and primershave been premixed (20) to a concentration of 18 M foreach primer and 5 M for the probe (ABI) in a final volumeof 10 l. All the reactions were obtained in a duplex PCRreaction with-actin (ACTB) as endogenous control (VIC

MGB Probe; ABI) at these conditions: 5 l TaqMan UniversalMaster Mix (ABI), 0.5 l 20 Assay Mix primer, 0.35 l 20

Assay Mix ACTB, and 3.5 l Tris-EDTA (TE). Reaction wasrun at 50C for 2 min, 95C for 10 min, and then at 40 cyclesat 95C for 15 s and 60C for 1 min. Data were analyzed usingthe standard curve method. The relative quantities of target

transcripts were calculated from triplicate samples after nor-malization of the data against the housekeeping gene -actin.

Western blotting

Western blotting was performed running 20 40 g proteinon a 10 or 12% sodium dodecyl sulfate (SDS) gel (Invitrogen)and transferring it to a nitrocellulose membrane (pore size0.45 m; Whatman GmbH, Dassel, Germany). Membranes

were incubated with the primary antiphosphorylated-LKB1

(pLKB1) antibody (Santa Cruz Biotechnology, Inc., SantaCruz, CA, USA), which recognizes the LKB1 phosphorylatedat Ser-431, at a concentration of 1:500; with the anti-Ca 2/calmodulin-dependent protein kinase kinase (CaMKK) an-tibody (1:200, Santa Cruz); and with the anti-CaMKK anti-body (1:200, Santa Cruz) in 5% nonfat dry milk in Tris-buffered saline-Tween or in 5% BSA in Tris-buffered saline-Tween for CaMKK, overnight at 4C. Goat anti-rabbit IgGIRDye 800CW, 1:10000, (Li-COR Bioscience, Lincoln, NE,USA) was used as secondary antibody for pLKB1 andCaMKK, and donkey anti-goat IgG IRDye 800CW, 1:10000,(Li-COR) as secondary antibody for CaMKK. After strippingthe membrane, the glyceraldehyde 3-phosphate dehydroge-nase (primary antibody 1:200, Santa Cruz; secondary goatanti-rabbit antibody 1:10,000, Li-COR) antibody was used to

normalize for equal loading. Band intensities were detectedwith Li-COR, and densitometry values were calculated withthe Image J program (U.S. National Institutes of Health,Bethesda, MD, USA).

Basal and insulin-mediated glucose uptake in humanadipocytes

Basal and insulin-mediated glucose uptake was performed asdescribed previously (23). On day 1, the differentiationmedium of the human adipocytes was removed. Cells were

washed 3 in warm PBS and kept in DMEM/F12 containing5 mM glucose and 3% FBS. Supplements were added on day2. On day 3, at t 0 min, 100 nM insulin was added to half

of the cells. At t 20 min, 1 C 2-deoxy-d-[3H(G)] deoxy-glucose (Perkin Elmer, Boston, MA, USA) was added to all

wells. After another 15 min, the cells were washed 3 inice-cold PBS and lysed in 0.1% SDS. Radioactivity was mea-sured in a scintillation counter.

Lipid staining

Rat liver samples were stained with Oil-Red-O. Staining ofsections was scored for droplet distribution and for stainingintensity in positive cells as follows: 1) distribution: universal(all or almost all cells positive), score 2; zonal (groups of cells

with intervening negative areas), score 1; negative (only theodd cell or no cells positive), score 0; 2) staining intensity in

positive cells: strong (mixture of large and small globules offat per cell), score 2; weak (only small globules of fat), score 1.The analysis was performed by adding the scores of1 and 2.

Endocannabinoid content

Whole hypothalamus was homogenized and extracted with achloroform/methanol method, and extracts were dried un-der nitrogen stream. Levels of anandamide, 2-arachidonyl-glycerol (2-AG) and 1-AG were quantified by liquid chroma-tography/inline mass spectrophotometry, as described (24).The amount of anandamide and 2-AG in the samples wasdetermined by using inverse linear regression of standard

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curves. Values of 2-AG are expressed as femtomole or pico-mole per milligram wet tissue.

Hormone and biochemical measurements in the rat model

Corticosterone and ACTH were measured as described pre-viously (25). Insulin, leptin, total (both acylated and unacy-lated) ghrelin, and adiponectin (Linco, St. Louis, MO, USA)and glucose, triglycerides, and cholesterol (Instrumental Lab-oratory, Lexington, MA, USA) were measured using commer-cial assays. The homeostasis model assessment (HOMA)index was calculated by multiplying insulin (U/ml) andblood glucose (mmol/L), divided by 22.5.

Statistical analysis

Studentsttest or 1-way analysis of variance with least squaredifference for post hoccomparisons was applied for normallydistributed data, whereas the Mann-WhitneyU and Kruskal-

Wallis tests were used for non-normally distributed data.Correlations were carried out using Spearmans rank corre-lations. P 0.05 was considered to indicate statistical signif-icance. Data are shown as means seunless stated otherwise.

RESULTS

Rat model of Cushings syndrome

We have created a rat model of Cushings syndromeaccording to the model described by Dallmans group(16, 17). Biochemical and hormonal parameters con-firmed that glucocorticoid-treated rats had the ex-pected hormonal and metabolic profile of Cushingssyndrome. In glucocorticoid-treated rats, corticoste-

rone levels were higher (P0.001) and ACTH levelswere lower (P0.007), compared with the 2 controlgroups (Fig. 1A,B). Insulin concentrations were higherin corticosterone-treated rats compared with the 2other groups (P0.002), with no difference in glucoseconcentrations (Fig. 1C, D). The HOMA-index forcorticosteroid-treated rats (2.20.3) indicates insulinresistance, whereas the index was normal in the 2control groups (0.90.07 and 0.80.1). Plasma leptin

levels were higher in the corticosterone-treated ratscompared with the 2 other groups (P0.005, Fig. 1E).In contrast, we found no difference in either adiponec-tin or ghrelin levels between the 3 groups (data notshown).

Corticosteroid-treated animals had significantlyhigher plasma total cholesterol levels compared withthe 2 control groups (P0.003, Fig. 1F). Triglyceridelevels were higher in both groups of sucrose-drinkinganimals treated with either placebo or corticosterone(Fig. 1G). The more pronounced effect of corticoste-rone per se on hypercholesterolemia compared withhypertriglyceridemia is concordant with data reported

in Cushings syndrome in humans (26). Total calorieintake was higher in the 2 sucrose-drinking groupscompared with saline-drinking animals (P0.001), andthere was a trend for higher calorie intake in corti-costerone-treated animals drinking sucrose com-pared with sucrose alone (P0.07, Fig. 1H). Caloricefficiency (grams gained/calorie ingested), in accor-dance to the marked catabolism of corticosteroid-treated animals, was lowest in the corticosterone-treated animals (P0.001).

Figure 1. Biochemical and hormonal pa-

rameters in plasma in rats drinking su-crose and treated with corticosterone(Cort) compared with rats treated withcholesterol pellets and drinking sucrose(Sucr) or saline (NaCl): A) corticoste-rone; B) ACTH; C) insulin; D) glucose;

E) leptin; F) total cholesterol; G) triglyc-eride levels; and H) total caloric intake(both chow and sucrose calories in-gested) over the experimental period.*P 0.05, **P 0.01, ***P 0.001; allcomparisons between the Cort group vs.

both Sucr and NaCl controls, unless shown otherwise; asterisks above Cort column indicate significant differencevs.both Sucr and NaCl groups.

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Adipose tissue

Rat model

Visceral white adipose fat depot weight was higher incorticosterone-treated rats (P0.01) compared withthe other 2 groups, whereas the inguinal fat depot, as ameasure of subcutaneous fat, was similar between cor-ticosteroid-treated rats and sucrose-drinking control

rats (Fig. 2A, B). In the visceral fat of corticosterone-treated rats, AMPK activity was significantly lower com-pared with the 2 control groups (P0.008). In contrast,in subcutaneous fat there was no difference in AMPKactivity between the 3 animal groups (P0.16, Fig.2C,D). PEPCK and HSL mRNA expression were higherin the visceral adipose tissue of corticosteroid-treatedanimals compared with the other 2 groups (P0.01,0.006), and FAS and SREBP1c mRNA were higher incorticosteroid-treated rats compared with saline-drink-ing rats (P0.03, 0.02, Fig. 2E). By contrast, we foundno difference between these gene products in subcuta-neous adipose tissue except for HSL (P0.003), sug-

gesting an influence of corticosteroids on gluconeogen-esis and lipogenesis primarily in visceral but not insubcutaneous fat tissue. The predominant AMPK sub-unit in adipose tissue, AMPK1, showed no differencein visceral adipose tissue mRNA expression between thegroups (Fig. 2F), whereas the expression level of the2-subunit was negligible. We also studied the expres-sion of the AMPK-regulating enzymes pLKB1 as well asCaMKK and CaMKK with Western blotting. Thevisceral fat tissue of glucocorticoid-treated animalsshowed a reduced expression of CaMKK (69.25.4%of saline control,P0.05, Fig. 2G), compatible with thereduced activity of AMPK in this tissue. No significant

differences were detected for CaMKK or LKB1.

Human adipocytes

We also were able to show a decrease in AMPK activityin human adipocytes treated with dexamethasone 1 Mfor 24 h compared with vehicle-treated adipocytes(P0.04, Fig. 2H), suggesting a direct cellular effect ofcorticosteroids on adipocyte AMPK activity. The dose-response curve with metformin 0.1 to 10 mM showedthat metformin at doses of 1 and 10 mM significantlyinduced AMPK activity in human adipocytes comparedwith control. The inhibitory effect of dexamethasone

on AMPK activity was antagonized by coadministrationof metformin 10 mM, which increased AMPK activity to224 14% compared with dexamethasone treatmentalone (P0.01, Fig. 2I). We studied the downstreamenzyme FAS in response to metformin and dexa-methasone. Similar to in vivo rat visceral adiposetissue, dexamethasone 1 M for 24 h significantlystimulated FAS expression (17430% of control,P0.05, Fig. 2J). Metformin (10 mM) treatment onits own inhibited FAS expression (6311%), and thisinhibition was abolished by dexamethasone (met-formin vs. dexamethasonemetformin, P0.001).

We furthermore studied the effect of dexamethasoneon glucose uptake in human adipocytes (Fig. 2K). Basalglucose uptake was inhibited by dexamethasone(771% of control, P0.05) and stimulated by met-formin (23110% of control,P0.001), whereas coad-ministration of dexamethasonemetformin decreasedbasal glucose uptake compared with metformin alone(P0.05). Similar results were obtained when insulin-induced glucose uptake was studied. Dexamethasoneinhibited (890.7% of insulin alone,P0.05), whereasmetformin further stimulated, insulin-induced glucoseuptake (1762% of insulin alone,P0.001). Coadmin-istration of dexamethasonemetformin decreased in-sulin-stimulated glucose uptake compared with met-formin alone (P0.05).

Liver

To confirm the metabolic effects of corticosteroids onrat liver, we first performed lipid staining of sections ofrat livers. Corticosterone-treated rats showed a higherliver lipid content compared with placebo-treated rats

drinking either sucrose or saline (P0.002, Fig. 3A).Higher FAS mRNA was found in corticosteroid-treatedrats compared with the 2 control groups (P0.001),whereas expression of PEPCK, SREBP1c, and glucose-6-phosphatase (G6P) was not significantly different(Fig. 3B).

However, contrary to our initial expectations, AMPKactivity in the liver was significantly higher in cortico-sterone-treated rats compared with the other 2 groups(P0.01, Fig. 3C). To investigate whether the observedeffect was direct or indirect, we used a human hepa-toma cell line. Here, similar to the rodentin vivodata,AMPK activity was increased following dexamethasone

1 M treatment after 6 h compared with control cellcultures (P0.001, Fig. 3D), whereas at 24 h no signif-icant changes were observed.

AMPK1-subunit mRNA expression was lower in thecorticosteroid-treated animals compared with the sa-line-drinking rats (P0.01), whereas no difference wasobserved between corticosteroid-treated and sucrose-drinking animals in AMPK2-subunit expression be-tween the 3 groups (Fig. 3E).

Hypothalamus

Humans with Cushings syndrome demonstrate in-

creased appetite (4), and we therefore studied theinfluence of corticosteroids on AMPK activity in thehypothalamus. We observed a higher AMPK activity incorticosterone-treated rats compared with sucrose-drinking control rats (P0.001). The lower AMPKactivity in sucrose-drinking compared with saline-drink-ing control rats confirmed the inhibitory effect ofsucrose on hypothalamic AMPK activity. Corticosteroneadministration counteracted the effects of sucrose andincreased hypothalamic AMPK activity to levels compa-rable to saline-drinking animals (Fig. 4A). These arecompatible with an increase in appetite and, as noted



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Figure 2.Weight of adipose tissue depots in rats drinking sucrose and treated with corticosterone (Cort) rats compared with ratstreated with cholesterol pellets and drinking either sucrose (Sucr) or saline (NaCl): visceral fat depots (A), subcutaneous(white) adipose tissue depots (B). Effects of glucocorticoid treatment on adipose tissue: AMPK activity in rat visceral adiposetissue (C); AMPK activity in rat subcutaneous adipose tissue (D); real-time RT-PCR (PEPCK, HSL, FAS, and SREBP1c) in rat

visceral and subcutaneous adipose tissue (E); real-time RT-PCR for AMPK1 subunit in rat visceral adipose tissue (F); CaMKKprotein expression in rat visceral adipose tissue (G); AMPK activity in human adipocytes treated with 1 M dexamethasone(Dexa) for 24 h compared with control treatment (expressed as % of control) ( H); AMPK activity in human adipocytes treated

with 1 M dexamethasone and 1 M dexamethasone plus 10 mM metformin (Met) for 24 h (I); FAS mRNA expression inhuman adipocytes treated with 1 M dexamethasone, 10 mM metformin, and 1 M dexamethasone plus 10 mM metformin for24 h (J); and basal (left panel) and insulin-induced (right panel) glucose uptake in human adipocytes treated with 1 Mdexamethasone and 1 M dexamethasone plus 1 mM metformin for 24 h (K). *P 0.05, **P 0.01, ***P 0.001; allcomparisons between the Cort group vs. both Sucr and NaCl controls, unless shown otherwise; asterisks above Cort columnindicate significant difference vs. both Sucr and NaCl groups.



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Figure 3.Effects of glucocorticoid treatment on liver:A) lipid staining;B) RT-PCR of FAS, SREBP1c, PEPCK, and G6P; C) AMPKactivity in rat liver; D) AMPK activity in human hepatoma cell line treated with 1 M dexamethasone for 6 h compared withcontrols (expressed as % of control); and E) real-time RT-PCR for AMPK subunits.

Figure 4. Effects of glucocorticoid treatment on hypothalamus: A) AMPK activity in rat hypothalamus; B) endocannabinoid(2-AG) content in hypothalamus;C) AMPK activity in primary hypothalamic cultures treated with 1 M dexamethasone for 6 hcompared with control treatment (expressed as % of control); and D) AMPK activity in primary hypothalamic cultures treated

with 1 M dexamethasone for 24 h compared with treatment with 1 M dexamethasone and 1 mM metformin for 6 h.



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above, we observed an increase in total calorie intake incorticosterone-treated compared with sucrose-drinkingrats (Fig. 1H).

To explore possible mechanisms as to how cortico-steroids increase hypothalamic AMPK activity, we mea-sured the endocannabinoid content in the 3 groups ofanimals. Similar to the pattern of the AMPK activity, theendocannabinoid 2-AG content was higher in cortico-sterone-treated rats than in sucrose-drinking controlrats (P0.01 Fig. 4B). Anandamide levels were alsohigher in corticosterone-treated rats than in sucrose-drinking control rats (P0.04, data not shown).

Primary rat hypothalamic cell cultures showed asignificant increase of the AMPK activity after treatmentwith dexamethasone 1 M for 6 h (P0.02, Fig. 4C).Addition of 1 mM metformin inhibited the effect ofdexamethasone and decreased AMPK activity to 26 2% of the level seen with dexamethasone alone(P0.01, Fig. 4D).

Skeletal and heart muscle

We found no difference in total AMPK activity in eitherwhite or dark muscle between the 3 groups. We there-fore analyzed 1 or 2 AMPK activity separately, butagain no changes were detected (data not shown). Inthe heart, AMPK activity was lower in corticosterone-treated rats compared with the other 2 groups(P0.001,Fig. 5).

DISCUSSION

Glucocorticoid excess leading to Cushings syndrome isa common problem, caused by either endogenous

hypercortisolism or, more frequently, by exogenousglucocorticoid administration for a wide range of dis-eases. In this study, we have speculated that many of thedetrimental effects, especially those related to the met-abolic syndrome, might be mediated at least partially byglucocorticoid-induced changes in AMPK activity. Ourresults show that corticosterone changes AMPK activityand some of its downstream targets in various tissues ina tissue-specific manner that may explain the increase

in appetite, accumulation of lipids in visceral adiposetissue, the development of fatty liver, and some of thecardiac effects of Cushings syndrome.

Corticosterone-treated animals had significantly in-creased corticosterone and decreased ACTH levels,became markedly hyperinsulinemic and hyperleptine-mic, showed high triglyceride and cholesterol levels,and developed visceral obesity. In rats, it is well knownthat chronic stressors associated with corticosteroneexcess usually decrease chow intake (16). This result isin contrast to humans, who generally demonstrateweight gain and increased appetite in response toexcess glucocorticoids or chronic stress (27). To coun-teract the effect of glucocorticoids on catabolism, in themodel of Cushings syndrome established by Dallman etal. (16, 17), 30% sucrose is added to the diet ofcorticosterone-treated rats. In this model, consideredone of the best animal models to study chronic excessglucocorticoid action and the consequent insulin resis-tance, we found a significantly increased appetite in theglucocorticoid-treated rats compared with saline-treated rats and a strong trend compared with sucrose-

drinking rats. The palatability of the sucrose could bepart of the increased total calorie intake in the sucrose-treated animals (16).

Typically, with excess glucocorticoid activity, there isa detrimental shift from the more inert subcutaneousfat to accumulation of the metabolically more disadvan-tageous visceral, intra-abdominal fat (5). Glucocorti-coid effects on adipose cell differentiation and lipidaccumulation are more pronounced in visceral than insubcutaneous fat, suggesting that glucocorticoids mayplay a pivotal role in the pathogenesis of central obesity(28). The higher level of local production of activeglucocorticoids from inactive metabolites by levels of

visceral adipose tissue 11-hydroxydehydrogenase-1 inobesity and data from the tissue-specific 11-hydroxy-dehydrogenase-1 knockout mice also support an impor-tant role of glucocorticoids in visceral adiposity (29).Here we show several changes demonstrated in re-sponse to glucocorticoids by visceral but not by subcu-taneous fat. AMPK activation in adipose tissue inhibitslipogenesis and lipolysis and stimulates lipid oxidation.Thus, inhibition of AMPK leads to increased lipid storesin association with enhanced lipolysis, leading to therelease of free fatty acids (30). AMPK activity in ourexperiment was significantly decreased in visceral butnot in subcutaneous adipose tissue compared with

controls. Therefore, we suggest that this is at least oneof the mechanisms for the prominent effect of cortico-steroids in accelerating lipid deposition in visceraladipose tissue, as these effects correspond to the effectsof AMPK on lipid-regulating enzymes. To study themechanism of the AMPK activation, the expression ofthe upstream kinases LKB1, CaMKK, and CaMKKwas determined. Visceral adipose tissue showed de-creased expression of CaMKK, and we speculate thatthis could explain, at least in part, the reduced AMPKactivity measured. Our data suggest that corticoste-roids,viaa decrease in AMPK activity, increase the gene

Figure 5. Effects of corticosterone treatment on AMPKactivity in rat heart muscle.



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expression of fatty-acid synthesis enzymes (FAS,SREBP1c, and PEPCK) and HSL, which increases therelease of lipids into the circulation. This change doesnot exclude an additional direct glucocorticoid effectviathe nuclear glucocorticoid receptor on the expres-sion of some of these enzymes (31, 32). Similar to theinvivorat data, in vitroincubation of human adipocyteswith dexamethasone led to a fall in AMPK activity,indicative of a direct effect of glucocorticoids on hu-man adipocyte AMPK. AMPK has a well-described effecton glucose uptake, and we observed that dexametha-sone reduced both basal and insulin-induced glucoseuptake in human adipocytes. The 1-subunit is thepredominant subtype in adipose tissue, but there wasno change in its mRNA expression with glucocorticoidtreatment. Thus, these data suggest that the phosphor-ylation of AMPK, but not the total level of AMPK, isinhibited by treatment with glucocorticoids.

Glucocorticoid hormones and drugs are also knownto cause abnormal metabolism in the liver, and we haveshown previously that Cushings syndrome in humans isassociated with increased hepatic fat accumulation

(33). Accordingly, lipid staining revealed a higher fatcontent in the livers of corticosteroid-treated rats com-pared with controls. Unexpectedly, hepatic AMPK ac-tivity was significantly higher in corticosteroid-treatedrats compared with sucrose-treated controls. On theother hand, previous reports in rats and mice subcuta-neously injected with dexamethasone for 5 days, as wellas in primary cultured hepatocytes, have also shownsignificant up-regulation of hepatic AMPK expressionby glucocorticoids (34), therefore supporting our find-ings. Acute hepatic overexpression of constitutively-active AMPK leads to a significant accumulation oflipids within the liver (35), as gluconeogenesis is sup-

pressed and lipid oxidation is facilitated (36); theresultant shift to lipid oxidation as a source of fuelinduces a net flux of fatty acids into the liver (35). As wealso have shown here, the decreased AMPK activity invisceral fat and the possible consequent increase ingenes involved in fat metabolism could lead to a shift toenhanced lipogenesis as well as lipolysis from adiposetissue, resulting in an efflux of fatty acids from theadipose depots to the liver and thus to increasedhepatic lipid uptake. The increased flux of centripetalhepatic lipid delivery may account for the hepaticsteatosis characteristic of Cushings syndrome (33). Inthe model of liver-specific acute overexpression of

constitutively active AMPK, total adipose tissue fell aslipids were transferred to the liver (35), but in Cush-ings syndrome this decrease would be counteracted bythe fall in adipose tissue AMPK and thus increase localadipose tissue lipogenesis. Hypercortisolism is knownto lead to increased lipolysis (37, 38). Therefore, glu-cocorticoid agents have a dual effect on lipogenesis andlipolysis in adipose tissue, but, as shown from theincreasing visceral fat depot mass, the effect on lipo-genesis prevails.

On the other hand, corticosteroid excess leads to astate of insulin resistance, as was indeed seen in this

model, and which is also characteristic of Cushingssyndrome (26). Insulin resistance could be due to theincrease in circulating free fatty acids consequent toincreased fat tissue lipolysis and would be itself respon-sible for the increased liver lipid accumulation. Glu-cocorticoids also have been shown to impair insulinsignalingviareduced cellular content of insulin recep-tor substrate (IRS-1) and reduction in tyrosine phos-phorylation in IRS-1 (3941). In agreement with pre-vious findings in primary cultured rat hepatocytes (34),we found that dexamethasone treatment significantlyincreased AMPK activity in a human hepatoma cell line,supporting ourin vivodata.

Obesity in Cushings disease results in part fromincreased food intake due to central nervous systemeffects (4, 4244), a complex process that is controlledby both hypothalamic and peripheral factors, andAMPK has been shown to play an important role(913). Administration of glucose inhibits hypotha-lamic AMPK activity (11), and we therefore analyzed 3groups of animals, with the saline-drinking group beingan appropriate control for the influence of sucrose on

hypothalamic AMPK activity. Sucrose inhibited hypo-thalamic AMPK activity, as shown by a significantlylower hypothalamic AMPK activity in sucrose-drinkingcompared with saline-drinking control rats. Corticoste-rone administration counteracted the sucrose effectsand increased hypothalamic AMPK activity to levelscomparable to saline-drinking animals. This increase inhypothalamic AMPK activity would lead to an increasedtotal calorie intake. Because of their marked catabo-lism, the experimental rats did not increase their netweight, despite the increase in adipose tissue weight.

Hypothalamic slices treated in vitro with dexametha-sone showed an increase in endocannabinoid levels viaa

rapid, membrane receptor-dependent mechanism (14,15), whereas chronic glucocorticoid treatment has beenreported to increase the 2-AG content of the rat amygdala(45). Here we show that glucocorticoid treatment in-creases hypothalamic 2-AG contentin vivo. As it has beenshown that cannabinoids have a direct effect on hypotha-lamic AMPK activity (13), this relation provides a possiblemechanism for the orexigenic effect of glucocorticoids.We therefore suggest that glucocorticoids increase appe-titeviaendocannabinoid mediation in the hypothalamus,although the involvement of other mediators, such asneuropeptide Y, agouti-related peptide, and pro-opi-omelanocortin, is also possible (46, 47).

Metformin is a mainstay of therapy in the treatmentof type 2 diabetes. The ability of metformin to lowerblood glucose requires LKB1 and AMPK signals (48,49). In our study, metformin reversed the effects ofcorticosteroids on AMPKin vitroboth in primary hypo-thalamic cell culture as well as in adipocytes, suggestingthat metformin and glucocorticoids influence theAMPK signaling pathway in opposite ways and that themetformin effect is able to override the cortisol effect.In addition, metformin treatment was able to reversethe effects of dexamethasone on 2 AMPK-regulatedprocesses in human adipocytes: FAS mRNA expression



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Received for publication July 17, 2007.Accepted for publication December 6, 2007.


ampk regulada por glucocorticoides

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