Life Sciences 82 (2008) 1262-1271

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Citrate diminishes hypothalamic acetyl-CoA carboxylase phosphorylation andmodulates satiety signals and hepatic mechanisms involved in glucosehomeostasis in rats

Maristela Cesquini a, Graziela R. Stoppa a, Patrícia O. Prada b, Adriana S. Torsoni c, Talita Romanatto b,Alex Souza c, Mario J. Saad b, Licio A. Velloso b, Marcio A. Torsoni b,c,⁎a Departamento de Bioquímica, IB, Universidade Estadual de Campinas, SP, Brazilb Departamento de Clínica Médica, Universidade Estadual de Campinas, CP6109, CEP13.083-970, Campinas, SP, Brazilc Universidade Braz Cubas, CEP 08.773-380, Mogi das Cruzes, SP, Brazil

⁎ Corresponding author. Universidade Braz Cubas,Francisco Rodrigues Filho, 1233. Mogilar, Mogi das CTel.: +55 11 4791 8000; fax: +55 19 37888950.

E-mail address: torsoni@yahoo.com (M.A. Torsoni).

0024-3205/$ – see front matter © 2008 Elsevier Inc. Aldoi:10.1016/j.lfs.2008.04.015

A B S T R A C T

A R T I C L E I N F O

Article history:

The hypothalamic AMP-acti Received 17 January 2008Received in revised formAccepted 22 April 2008

Keywords:HypothalamusInsulinCitrateLiverAcetyl-CoA carboxilaseAMPK

vated protein kinase (AMPK)/acetyl-CoA carboxylase (ACC) pathway is known toplay an important role in the control of food intake and energy expenditure. Here, we hypothesize that citrate,an intermediate metabolite, activates hypothalamic ACC and is involved in the control of energy mobilization.Initially, we showed that ICV citrate injection decreased food intake and diminished weight gain significantlywhen compared to control and pair-fed group results. In addition, we showed that intracerebroventricular(ICV) injection of citrate diminished (80% of control) the phosphorylation of ACC, an important AMPK substrate.Furthermore, citrate treatment inhibited (75% of control) hypothalamic AMPK phosphorylation during fasting.In addition to its central effect, ICV citrate injection led to lowblood glucose levels during glucose tolerance test(GTT) and high glucose uptake during hyperglycemic–euglycemic clamp. Accordingly, liver glycogen contentwas higher in animals given citrate (ICV) than in the control group (23.3±2.5 vs. 2.7±0.5 μg mL−1 mg−1,respectively). Interestingly, liver AMPK phosphorylation was reduced (80%) by the citrate treatment. Thepharmacological blockade of β3-adrenergic receptor (SR 59230A) blocked the effect of ICV citrate and citrateplus insulin on liver AMPK phosphorylation. Consistently with these results, rats treated with citrate (ICV)presented improved insulin signal transduction in liver, skeletalmuscle, and epididymal fat pad. Similar resultswere obtained by hypothalamic administration of ARA-A, a competitive inhibitor of AMPK. Our results suggestthat the citrate produced by mitochondria may modulate ACC phosphorylation in the hypothalamus,controlling food intake and coordinating a multiorgan network that controls glucose homeostasis and energyuptake through the adrenergic system.

© 2008 Elsevier Inc. All rights reserved.

Introduction

Enzyme AMP-activated protein kinase (AMPK) acts as a cellenergetic status sensor (Kahn et al., 2005; Hardie et al., 2006). Itresponds to and integrates nutrient and hormonal signals involved inenergetic homeostasis, thus modulating cellular function according toenergy availability. Once activated, AMPK inhibits fatty acid biosynth-esis through the phosphorylation of acetyl-CoA carboxylase (ACC) atits Ser79 residue. Whenever active, ACC catalyzes the formation ofmalonyl-CoA, which allosterically inhibits the entrance of long-chainacyl-CoA into the mitochondria to undergo β-oxidation for energyproduction (Long and Zierath, 2006). Initial studies have explored the

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multiple facets of AMPK activity in cells of peripheral tissues,especially in skeletal muscle (Fisher et al., 2002), where it is knownto promote glucose uptake through insulin-independent mechanism,and in the liver, where it plays a role in the control of glucoseproduction during fasting (Koo et al., 2005).

Some recent studies have evaluated the roles of AMPK in the controlof hypothalamic functions. In the hypothalamus, AMPK is highlyactivated during fasting or in the presence of orexigenic peptides suchas ghrelin and AGRP (Hardie, 1989; Kahn et al., 2005; Lee et al., 2005).After feeding or following the intracerebroventricular (ICV) adminis-tration of leptin and insulin, AMPK is inhibited (Minokoshi et al., 2004;Kim and Lee 2005; Lee et al., 2005). Once activated in thehypothalamus, AMPK drives to an orexigenic response, which isattenuated by feeding and by the subsequent increase in leptin andinsulin levels. Thus, in the hypothalamus, AMPK participates in thecomplex network that controls the feeding and fasting cycles (Longand Zierath, 2006). Recent studies have suggested that besides its

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direct role in energy acquisition by feeding control, hypothalamicAMPK might also play an indirect role in the control of peripheralenergy stores by controlling the hypothalamus-sympathetic nervoussystem axis (Kahn et al., 2005; Xue and Kahn 2006). For example, theadministration of leptin into ventromedial hypothalamus increasedheart, brown adipose tissue (BAT), and skeletal muscle glucose uptakethrough the mediation of a β-adrenergic mechanism (Haque et al.,1999). Furthermore, it has been demonstrated that systemic andintracerebroventricular administration of leptin increases sympatheticactivity to BATand other peripheral tissues (Collins et al., 1996; Hayneset al., 1997) and increases insulin sensitivity (Barzilai et al., 1997; Shiet al., 1998). As well as leptin, other nutrient factors such as pyruvate,glucose, and synthetic compounds (AICAR and sodium azide) arecapable of modulating hypothalamic AMPK and restoring ATP levels inneuronal cells (Lee et al., 2005).

Recently, Roman et al. (2005) showed that citrate promotes satietysignal when injected into the hypothalamus. Citrate is an intermediatemetabolite produced in the mitochondria in the citric acid cycle. It istransported to the cytoplasm from mitochondria and used in lipidbiosynthesis in response to an increase in the ATP/AMP ratio. Recently,a novel sodium-coupled citrate transporter (NaCT) has been describedin mouse brain (Inoue et al., 2002). It is expressed in most brainneurons. Furthermore, citrate is a positive allosteric effector of theacetyl-CoA carboxylase activity and a negative modulator of phospho-fructokinase, a key glycolysis pathway enzyme. Supported by the factthat leptin, insulin, glucose, and pyruvate inhibit AMPK, promotesatiety signal, and increase peripheral glucose uptake, it wasinteresting to study the role of citrate in the neuronal and peripheralmechanisms of energy acquisition and disposal. In addition, theidentification of the target protein would be interesting for thedevelopment of new drugs that act in the hypothalamus, as they couldmodulate satiety and glucose homeostasis.

To address this issue, we performed a series of experiments in ratsafter citrate intracerebroventricular injection treatment. To obtainhigh hypothalamic AMPK activity, the animals were fasted. Using thisapproach, we demonstrated that citrate can diminish hypothalamicAMPK/ACC phosphorylation and reduce food intake. It can alsoincrease glucose uptake and glycogen level in the liver and reduceliver AMPK phosphorylation. Besides, upon central administration,citrate can improve insulin signal transduction in the liver and isdelivered, at least in part, by sympathetic β-adrenergic signals.

Materials and methods

Experimental animals and surgical procedures

Male Wistar Hannover rats (12 wk old, 250–280 g) from theUniversidade Estadual de Campinas animal breeding center wereused in all experiments. The rats were maintained at roomtemperature (25 °C) and in 12:12-h light–dark cycles with free accessto water and chow, unless otherwise indicated. The rats werechronically instrumented with an intracerebroventricular (ICV)cannula and kept under controlled temperature and light–darkconditions (07:00–19:00 h) in individual metabolic cages (BeiramarIndustria e Comercio LTDA). Seven days after ICV cannula installation,the rats were tested for cannula function and position and thereafterrandomly selected for one of the experimental groups. The generalguidelines established by the Brazilian College of Animal Experi-mentation were followed throughout the study. Briefly, the animalswere anesthetized with 50 mg/kg ketamine and 5 mg/kg diazepam(ip) and positioned onto a Stoelting stereotaxic apparatus after theloss of cornea and foot reflexes. A 23-gauge guide stainless steelcannula with indwelling 30-gauge obturator was stereotaxicallyimplanted into the lateral cerebral ventricle at pre-establishedcoordinates, anteroposterior, 0.2 mM from bregma; lateral, 1.5 mM;and vertical, 4.2 mM, according to a previously reported technique

(Michelotto et al., 2002). Cannulas were considered patent andcorrectly positioned by dipsogenic response elicited after injection ofangiotensin II (2 μL of solution 10−6 M) (McKinley et al., 2000). Allprocedures were approved by the Ethical Committee of theUniversidade Braz Cubas, Mogi das Cruzes, SP.

Biochemical and hormonal measurements

Plasma insulin was determined by RIA according to a previouslydescribed method (Scott et al., 1981). Serum glucose was determinedby the glucose oxidase method (Trinder, 1969). Plasma corticosteronewas determined by ELISA (DSL inc.) according to manufacturer'sdirections. Glycogen was measured in liver fragments in the morning,according to a previously described method (Pimenta et al., 1989).Briefly, digestion procedures were begun by adding 30% aqueous KOH(w/v) to each sample at 3-fold the tissue weight rate. The sampleswere then heated in shaking water bath at 100 °C for 20 min. Afterremoval from the water bath, the samples were vortexed for 30 s andchilled on ice for 5 min. After cooling, 200 μL of the digested tissueKOH solution and 200 μL of 95% ethanol were pipetted into vials. Thevials were then vortexed for 5 s and placed into shaking water bath at100 °C for 15 min. Next, 1.2 mL deionized water was added to eachsample, vortexed for 10 s, and allowed to stand at room temperaturefor 5 min. Following the digestion procedure, the samples were readyto be assayed in microwell plates with 96 individual well. Threereplicates of 25 μL of each standard and each sample were pipettedinto individual wells. Next, 10 μL of 80% aqueous phenol (v/v) and200 μL of reagent grade H2SO4 were added to plate wells. Themicrowell plate was covered, allowed to settle at room temperaturefor 5–10 min, and then placed in a Microplate Reader at 490 nm. Aserial dilution of a single high concentration glucose solution wasused for curve calibration. All blood samples were collected from thetail vein.

Citrate, ARA-A, SR 59230A and insulin treatment protocols

For evaluation of the citrate effect on the food intake and bodyweight, ICV-cannulated rats were ICV-treated with 2.0 μL of 10−2 Mcitrate (20 nmol) invariably at 18:00 h, during 8 days (see diagram). Toinsulin signal transduction, GTT and hyperinsulinemic–euglycemicclamp procedures ICV-cannulated rats were ICV-treated with 2.0 μL of10−2 M citrate (20 nmol) or AMP analog adenine 9-β-D-arabinofurano-side (ARA-A, competitive inhibitor of AMPK) (insulin signal transduc-tion) 30 min before the beginning of the experiments. Whennecessary, SR 59230A, a β3-receptor antagonist, was administeredby intraperitoneal injection (0.1 mg/kg body weight) 30 min beforecitrate (ICV).

For evaluation of the molecular events of the insulin signaltransduction pathway, 0.2 mL saline (0.9% NaCl), with or withoutinsulin (10−4 M), was injected through the cava vein immediatelybefore tissue extraction. Whenever necessary, food was withdrawn12 h before treatment. For tissue extraction, the rats were anesthe-tized and the hypothalamus and peripheral tissues were rapidlyremoved after different intervals as described under Results.

Tissue extraction, immunoprecipitation and immunoblotting

After specific treatments, the rats were anesthetized and decapi-tated. Tissues were obtained and homogenized in freshly preparedice-cold buffer (1% Triton X-100, 100 mM Tris, pH 7.4, 100 mM sodiumpyrophosphate,100mM sodium fluoride,10mMEDTA,10mM sodiumvanadate, 2 mM PMSF, and 0.01 mg aprotinin/mL). Insoluble materialwas removed by centrifugation (10,000 g) for 25 min at 4 °C. Aliquotsof the resulting supernatants containing 2.0 mg of total protein wereused for immunoprecipitation with specific antibodies [anti-IR(rabbit; sc-711), anti-IR substrate-1 (IRS-1; rabbit; sc-559), or anti-

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IRS-2 (goat; sc-1555)], all from Santa Cruz Biotechnology (Santa Cruz,CA), at 4 °C overnight, followed by the addition of protein A-Sepharose6MB (Pharmacia, Uppsala, Sweden) for 2 h. The pellets were washedthree times in ice-cold buffer (0.5% Triton X-100, 100 mM Tris, pH 7.4,10 mM EDTA, and 2 mM sodium vanadate) and then resuspended inLaemmli sample buffer and boiled for 5 min before separation in SDS-PAGE using a miniature slab gel apparatus (Bio-Rad, Richmond, CA).Electrotransfer of proteins from the gel to nitrocellulose wasperformed for 90 min at 120 V (constant). The nitrocellulose transferswere probed with anti-phospho-tyrosine from Santa Cruz Biotechnol-ogy (Santa Cruz, CA). The blots were subsequently incubatedwith 125I-labeled protein A (Amersham, Aylesbury, UK). After identification ofIR, IRS-1 and IRS-2 tyrosine phosphorylation, membrane stripping andre-probing protocols were employed. The same membranes wereused to quantify the level of proteins through re-probing with thesame antibody used in the immunoprecipitation protocol. For directimmunoblot analysis, 0.2 mg protein from hypothalamus extracts wasseparated by SDS-PAGE, transferred to nitrocellulose membranes, andblotted with specific antibodies, anti-phospho-AMPK and anti-phospho-ACC (Cell Signaling Technology), anti-phospho-AKT(SER473) and anti-phospho-tyrosine from Santa Cruz Biotechnology(Santa Cruz, CA). Subsequently the blots were incubated with 125I-labeled protein A (Amersham, Aylesbury, UK). The results werevisualized by autoradiography with preflashed Kodak XAR film.Band intensities were quantified by optical densitometry of developedautoradiographs (Scion Image software, ScionCorp).

Intraperitoneal glucose tolerance test (ipGTT)

Intraperitoneal glucose tolerance test was performed after over-night fast. The rats were anesthetized as described above. ICV-cannulated rats were ICV-treated with 2.0 μL of 10−2 M citrate(20 nmol). After 30 min, an unchallenged sample (time 0) wascollected soon after a glucose bolus (2.0 g/kg body weight) wasadministered into the peritoneal cavity. Blood samples were collectedfrom the tail at 5, 15, 30, 60, 90, and 120 min for the determination ofglucose and insulin concentrations.

Hyperinsulinemic–euglycemic clamp procedures

With this technique, a constant rate of insulin is infused (iv) toincrease the uptake of circulating glucose by insulin-sensitive tissuesand to inhibit endogenous glucose production by the liver. The declinein plasma glucose is prevented by a concomitant variable rate ofglucose infusion. The amount of exogenous glucose required tomaintain plasma glucose at its initial level is quantified by the GIR, ameasure of the ability of insulin to increase glucose uptake and tosuppress glucose production in a given subject, i.e., a measure of thesubject insulin sensitivity.

Firstly, after 5-h fasting, the ICV-cannulated rats were anesthetizedwith sodium pentobarbital (50 mg/kg body weight) injected ip, and

Scheme

catheters were then placed into the left jugular vein (for tracerinfusions) and carotid artery (for blood sampling), as previouslydescribed (Prada et al., 2000). Each animal was monitored for foodintake and weight gain for 5 days after surgery to ensure completerecovery. Food was removed 12 h before the beginning of in vivostudies. For these procedures, ICV-cannulated and catheterized ratswere ICV-treated with either 2.0 μL of 10−2 M citrate (20 nmol) orsaline. After 30 min a 120-min hyperinsulinemic–euglycemic clampprocedure was conducted in conscious and unrestrained rats, aspreviously described (Combs et al., 2001). A prime continuous insulininfusion at a rate of 3.6 mU/kg body weight per minute to raise theplasma insulin concentration to approximately 800–900 pmol/L wasperformed. Blood samples (20 μL) were collected at 5-min intervals forthe immediate measurement of plasma glucose concentration and10% unlabeled glucose was infused at variable rates to maintainplasma glucose at fasting levels. Insulin-stimulated whole-bodyglucose flux was estimated using a prime continuous infusion ofHPLC-purified [3-3H] glucose (10 μCi bolus, 0.1 μCi/min) throughoutthe clamp procedure (Rossetti et al., 1997). Blood samples (10 μL) werecollected before the start and during the glucose infusion period(every 30 min) for measurement of plasma insulin concentrations. Allinfusionswere performed using Harvard infusion pumps. At the end ofthe clamp procedure, the animals were killed by a sodium pento-barbital iv injection. In separate experiments, glucose turnover basalrates were measured by continuously infusing [3-3H] glucose(0.02 μCi/min) for 120 min. Blood samples (20 μL) were taken at100, 110, and 120 min after the start of the experiment to determineplasma [3H] glucose concentration.

Food intake and body weight measurements

The ICV-cannulated rats not previously treated were maintainedin individual cages with standard rodent chow and water ad libitumfor 2 days for adaptation. Then citrate (ICV) or saline (ICV) wasadministered immediately before the beginning of the dark cycle.Control (saline) and citrate-treated rat food intake and body weightmeasurements (next eight days) were obtained daily at 08:00 h. Atday 9 and day 10 (recovery period), food intake was evaluated inboth groups without citrate administration (see experimentalprocedure diagram).

Diagram:Schemes applied to each protocol (Schemes 1 and 2).

Data presentation and statistical analysis

All numerical results are expressed as means±SE of the indicatednumber of experiments. Blot results are presented as direct compar-isons of bands in autoradiographs and quantified by densitometryusing the Scion Image software (ScionCorp). Student's t test forunpaired samples and variance analysis (ANOVA) for multiplecomparisons were used for statistical analysis as appropriate. The

1.

Scheme 2.

Fig. 1. Effect of citrate ICV treatment on food intake and body weight. (A) Food intake(24 h) was evaluated after ICV injection of citrate (20 nmol) (●) or saline (○)immediately before nocturnal period (18:00 h) in rats fed ad libitum during 8 days. (B)Body weight gain in control rats (saline ICV) (○), citrate rats (citrate ICV) (●) and pair-fed group (saline ICV) (□). Data aremeans±SE, n=8 per group, ⁎Citrate group vs. controlgroup and citrate group vs. pair-fed, p≤0.05.

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post-hoc test (Tukey) was employed when required. The level ofsignificance was set at pb0.05.

Results

Food intake and body weight

Since intracellular citrate is known to control ACC activity andmalonyl-CoA levels, we decided to investigate whether this meta-bolite could affect food intake and weight gain. A protocol ofexperiments was adopted for this investigation. In this protocol,citrate (20 nmol) was administered daily and the cumulative foodintake (24 h) in the control group and the citrate group wasevaluated during the next 8 days (treatment phase). As can beobserved in Fig. 1A, the group that received citrate through ICVinjection presented a food intake of about 14.9±0.22 g/24 h, while inthe control group, food intake was 22.9±0.32 g/24 h. The measure-ments performed at day 9 and day 10 (recovery phase) wereobtained without previous injection of citrate to discard the toxiceffect of injected citrate. As can be observed in Fig. 1A, food intake atday 10 was similar to that of the control group.

Consistent with decreasing food intake, the eight-day chronic ICVcitrate treatment led to bodyweight loss. As can be observed in Fig.1B,the animals treated with ICV citrate daily (18:00 h) did not gainweight, while the control group gained 13% at the end of theexperimental protocol (5 g/day). On the other hand, the animals (pair-fed) fed the same amount of chow diet ingested by the citrate groupdaily presented a weight gain (3.2 g/day) similar to that of the controlgroup during the experimental period.

Hypothalamic ACC and AMPK phosphorylation

ICV citrate injection reduced the ACC phosphorylation by approxi-mately 80% comparatively to the control group. This effect was notaccompanied by a reduction in the ACC expression (Fig. 2A). Inaddition, a 75% reduction in the phosphorylation of hypothalamicAMPK, an upstream ACC kinase, was observed at 30 min after citrateadministration (20 nmol) without affecting its protein level (Fig. 2B).

Blood insulin and corticosterone levels

Basal insulin (before ICV citrate) and fasted glucose levels in bloodwere similar to values 30 min after ICV injection of citrate. Asignificant decrease in the corticosterone level was observed at30 min after treatment with citrate ICV when compared to the basallevel (Table 1).

ICV citrate improves sensitivity to insulin, glucose uptake and increasesglycogen content in liver

To assess the functional role of hypothalamic ACC in the whole-body metabolism, we performed glucose tolerance test (GTT) and

hyperinsulinemic–euglicemic clamp in ICV citrate-treated rats. Asshown in Fig. 3A and B, the animals treated with citrate presented asignificantly lower (50%) area under the glucose curve (AUCgli) and asignificantly lower (22%) area under the insulin curve (AUCins) thanthe control group did, suggesting a much more efficient glucoseuptake during the GTT. This effect was confirmed in peripheral tissues

Fig. 2.Hypothalamic ACC and AMPK phosphorylation in control and citrate-treated rats.(A) ACC phosphorylation and (B) AMPK phosphorylation in hypothalamus of control(saline) or citrate-treated fasted rats. Fasted rats were sacrificed 30 min after saline orcitrate ICV, 20 nmol. This is representative of two separate experiments in a total of 4rats/treatment. The blots were quantified using Scion Corp software. Data are means±SE. ⁎pb0.05, citrate vs. control (saline).

Table 1Glucose, corticosterone and insulin levels in serum from control animals (basal) andanimals treated with citrate ICV

[Serum glucose]mg dL−1

[Serum corticosterone]ng mL−1

[Serum insulin]pmol L−1

Basal(without citrate)

98±4 205±52 8.3±0.9

30 minafter citrate (B)

93±4 115±25* 8.9±1.5

Values are means±SE from 5 animals. There were no significant differences in glucoseand insulin levels for any of the times available. *p≤0.05, B vs. basal.

Fig. 3. Effect of citrate (ICV) in the glucose tolerance test. (A) Area under the glucosecurve (AUCgli) obtained from glucose tolerance test (GTT) and (B) area under insulincurve (AUCins) obtained from glucose tolerance test (GTT). The animals received saline(control) or citrate (20 nmol, ICV) and after 30 min, a glucose bolus was administered(2.0 g/kg body weight). Blood samples were collected from the tail at 5, 15, 30, 60, 90,and 120min for the determination of blood glucose and insulin concentrations. Data aremeans±SE, n=5. ⁎p≤0.05, citrate-treated rats vs. control rats.

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by hyperinsulinemic–euglicemic clamp. The glucose infusion rate(GIR) (Fig. 4A) necessary to clamp glycemia at fasting levels in thepresence of a constant insulin infusion (3.6 mU/kg body wt/min) washigher in rats treated with ICV citrate (28.6±0.8 mg kg−1 min−1) thanin their respective controls (19.3±0.2mg kg−1 min−1). Accordingly, theinsulin-stimulated whole-body glucose disposal rates (Fig. 4B) werealso significantly increased in rats treated with ICV citrate (39.5±3.1mg kg−1 min−1) when compared to their respective controls (29.2±2.2 mg kg−1 min−1). In this regard, insulin-induced reduction ofglucose output (Fig. 4C) was more evident in citrate-treated rats thanin the control group (control: 18.0±1.5; control plus insulin: 10.2±1.0;citrate: 18.7±0.8; citrate plus insulin: 6.1±0.5 mg kg−1 min−1). Theliver glycogen content was higher in citrate-treated rats than inanimals that received saline (23.3±2.5 vs. 2.7±0.5 μg mL−1 mg−1,respectively) (Fig. 5).

Insulin signal transduction and AMPK phosphorylation in the liver of ratstreated with citrate by ICV injection

To evaluate whether ICV citrate improves insulin signal transduc-tion in the liver, we determined IR, IRS1, IRS2 (Fig. 6A, B, and C) andAKT (Fig. 7A) expression and phosphorylation stimulated by insulin inthe liver of fasted rats 30 min after ICV citrate injection. Theexpression of IR, IRS1, IRS2 and AKT/PKB was not altered by thetreatments performed (insulin, citrate, insulin plus citrate, ARA-A,ARA-A plus insulin). However, in liver, epididymal fat pad and skeletal

Fig. 4. Glucose infusion rates (GIR), whole-body glucose disposal rate, and hepaticglucose output (HGO) in clamped state during euglycemic–hyperinsulinemic clamp.The animals received saline (control) or citrate (20 nmol, ICV) and after 30 min theexperiments, a euglycemic–hyperinsulinemic clamp was performed in overnight fastedrats to determine (A) glucose infusion rate (GIR) (⁎p≤0.01, citrate vs. control), (B) whole-body glucose disposal rates (⁎p≤0.5, citrate vs. control) and (C) hepatic glucose output.Data are means±SE, n=5. p≤0.5, ⁎⁎control plus insulin vs. control, #citrate plus insulinvs. citrate, +citrate plus insulin vs. control plus insulin.

Fig. 5. Liver glycogen content of fasted rats that received saline (control) or citrate(20 nmol, ICV) at 18:00 h. Data are means±SE, n=5. ⁎p≤0.5, citrate vs. control.

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muscle IR, IRS-1, IRS-2 (Fig. 6) and AKT (Fig. 7A) phosphorylationstimulated by intravenous insulin administration were improved byprevious treatment with citrate injection (Fig. 7B and C). To evaluatethe possibility of the citrate effect (ICV) having been promoted byhypothalamic AMPK inhibition, we administered ARA-A, a competi-tive inhibitor of AMPK, via ICV to ICV-cannulated rats. ARA-Awas ableto improve the phosphorylation stimulated by insulin (IP) of IR, IRS1,IRS2 (Fig. 6A, B, and C) and AKT/PKB (Fig. 7A, B, and C) in a mannersimilar to that observed for the citrate treatment. However, neithercitrate nor ARA alone altered either adipose or skeletal p-AKT, whichwas not true for liver p-AKT. In another set of experiments, the

adrenergic pathway was blocked by the administration of SR 59230A,a β3-antagonist (ip). This procedure was performed in ICV-cannulatedrats 30 min before citrate injection (ICV). In this model theadministration of SR 59230A blocked the effect of citrate on theinsulin-induced serine phosphorylation of AKT in the liver (Fig. 8A).

Citrate administration exerted no effect on liver AMPK expression(Fig. 8B). Nevertheless, liver AMPK phosphorylation was significantlylower (80%) in animals treated with either insulin (ip) or citrate (ICV).These effects were blocked by previous (30 min before citrate ICV)intraperitoneal administration of SR 59230A, a β3-antagonist (Fig. 8B).

Discussion

In the present study, we evaluated the role played by thehypothalamic ACC/AMPK pathway in the control of whole-bodyglucose homeostasis. To warrant the biological relevance of thephenomena studied herein, we decided to employ physiologicalmeans to modulate AMPK/ACC phosphorylation. To stimulatehypothalamic AMPK, the rats were submitted to fasting. Fooddeprivation increases neuronal levels of AMP while reducing ATPlevels. The increased AMP/ATP ratio is one of the most importantphysiological mechanisms involved in the activation of AMPK (Hardieet al., 2006). One well-characterized substrate of AMPK is acetyl-CoAcarboxylase (ACC). AMPK inactivates ACC by phosphorylation of itsserine residue (Ser79) (Hardie, 1989). Central administration of eitherleptin, or insulin, or glucose markedly improved the energy andglucose homeostasis (Shi et al., 1998; Haque et al., 1999; Minokoshi etal., 2004). A mechanism that may play a role in the connection ofhypothalamic AMPK function and feeding control is the oscillation ofintracellular levels of malonyl-CoA. Ruderman and colleagues (Ruder-man et al., 2003) proposed that allosteric activation of ACC by citrate,rather than a change in its phosphorylation state, may be responsiblefor the increased malonyl-CoA levels associated with food intakeinhibition (Hu et al., 2003). Recently, it was elegantly shown thatleptin activates hypothalamic ACC to inhibit food intake, concomi-tantly to AMPK inhibition (Gao et al., 2007). Physiologically, citrate is apositive allosteric modulator of ACC. Citrate is an organic acidgenerated during the metabolism of lipids, carbohydrates, andamino acids, which is present in CSF in similar concentrations as inthe plasma (Hoffmann et al., 1993). The concentration of this acid inCSF is dependent on the neuronal production, which suggests that itplays an important role in brain metabolism. Recently, a novelsodium-coupled citrate transporter (NaCT) that could facilitate thetransport of citrate into mouse brain neurons has been described(Inoue et al., 2002). Moreover, the administration of citrate throughICV injection showed that this organic acid is capable of inducing

Fig. 6. Insulin transduction signal in liver from overnight fasted rat that received saline(control), citrate (20 nmol, ICV) or ARA-A (2 nmol, ICV) at morning. Citrate and ARA-Awere administered 30min before insulin (4 nmol) injection (iv). A, IP with anti-IR and IBwith anti-α-Py; B, IP with anti-IRS1 and IB with anti-α-Py; and C, IP with anti-IRS2 andIB with anti-α-Py. Total expression was evaluated through immunoblotting with anti-IR, anti-IRS2 and anti-IRS1. Bars show quantification of p-IR (A), p-IRS1 and p-IRS2normalized by the total respective protein in liver. Data are means±SE, n=5. p≤0.05,⁎B vs. A, C vs A; ⁎⁎D vs. C, B and A; #F vs. E, B.

Fig. 7. AKT phosphorylation in liver (A), epididymal fat pad (B), and skeletal muscle (C)from overnight fasted rats that received saline (control), citrate (20 nmol, ICV) or ARA-A(2 nmol, ICV) 30 min before insulin (4 nmol) injection (iv). Bars show quantification ofp-AKT normalized by total AKT protein in tissue. Data are means±SE, n=5. p≤0.05, ⁎Band C vs. A; #D vs. A, B, C, and E; E vs A; +F vs. E, C, B, and A.

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anorexigenic signaling (Roman et al., 2005), suggesting that citratewas able to cross the neuron membrane. Because the ACC activity isrelated to the leptin and the insulin anorectic effect, the centraladministration of citrate is expected to diminish food intake. In fact,food intake was significantly reduced by the central administration ofcitrate. In addition to the anorectic effect, ACC and AMPK phosphor-ylation were reduced after ICV citrate. Thus, these results suggest that

Fig. 8. Effect of citrate and β3-antagonist (SR 59230A) on liver AKT (A) and liver AMPK(B) phosphorylation stimulated by insulin in fasted rats. Citrate (20 nmol) wasadministered 30 min before either saline or insulin injection (4 nmol, iv). Theintraperitoneal administration of SR 59230A (0.1 mg/kg body weight) was performed30 min before citrate (ICV). Bars represent p-AKT normalized by total AKT protein andp-AMPK normalized by total AMPK protein in liver. Data are means±SE, n=5. (p-AKT)p≤0.05, ⁎B vs. A, B vs C; C vs A, C vs E; ⁎⁎D vs. A, B, C, E, and F. (AMPK) p≤0.05, ⁎B, C,and D vs. A, E, and F; #E and F vs. B, C, and D.

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citrate improves the ACC activity by inhibiting the AMPK phosphor-ylation. The up-regulation of ACC would increase the malonyl-CoAformation in the brain, a phenomenon known to participate in thecontrol of feeding (Hu et al., 2003; Lam et al., 2005) by interactingwiththe recently identified brain isoform of CPT-1. Although citrate is aninhibitor of phosphofructokinase and therefore diminishes theproduction of ATP by the glycolytic pathway, the effect of citrate onAMPK/ACC is sufficiently robust. The mechanism through which ICVcitrate may mediate the AMPK inhibition has to be determined yet.

Stimuli that lead to increased AMPK activity promote food intake(Kim and Lee, 2005). During regular feeding and fasting cycle, theincreased AMP/ATP ratio achieved in fasting seems to be the mostimportant factor leading to AMPK activation. Conversely, increasedsubstrate availability and increased blood levels of leptin and insulinact in concert to inhibit AMPK activity during the fed state (Lee et al.,2005). The mechanism by which hypothalamic AMPK regulates foodintake remains unclear. Modulation of hypothalamic neurotransmitterexpression is one of the candidate mechanisms involved in thisphenomenon. For example, the treatment of mice with the chemicalactivator of AMPK activity, AICAR, increases the expression of NPY inarcuate nucleus (Kim et al., 2004). In addition, the expression ofconstitutively active AMPK in this anatomical site promotes an

increase in the expression of NPY and AgRP, while the expression ofdominantly negative AMPK leads to its decrease (Minokoshi et al.,2004). Evaluation of mRNA levels of NPY and POMC performed inhypothalamic extract through RT-PCR indicated that citrate treatmentincreased POMC and inhibited NPY expression (data not shown).These results corroborate the data obtained by Lee and colleagues (Leeet al., 2005), who showed increased AgRP expression after theactivation of AMPK, whereas the exposure of cells to high ATP levelsdecreased AMPK phosphorylation and AgRP expression in neuronalcell lines in culture. Thus, it seems that regulation of neurotransmitterexpression is an important mechanism involved in AMPK-dependentcontrol of feeding and a chemical agent capable of modulating both,AMPK and ACC activities, would be expected to promote a modulationin feeding. This seems to be the case of citrate when it is administeredvia ICV. In the presence of this acid, AMPK and ACC were lessphosphorylated, indicating a cell anabolic state. In this condition, thecytoplasm could present highmalonyl-CoA and ATP levels, resulting inan anorexigenic behavior.

Recent evidence suggests that the signals in the hypothalamusthat affect food intake are also transmitted to peripheral tissues toalter energetic and glucose homeostasis (Dowell and Cooke, 2002;Minokoshi and Kahn, 2003; Cha et al., 2005). Consistent with thefact that AMPK is modulated in response to nutrients and hormonalsignals, ACC is phosphorylated and inactivated by food withdrawal(Thampy and Wakil, 1988). The pivotal role of lipids and malonyl-CoA in glucose homeostasis and food intake has been widelydiscussed in studies performed with rats that received intracereb-roventricular injection of fatty acids, glucose, and pharmacologicalinhibitors of fatty acid synthase and ACC (Obici et al., 2002; Lamet al., 2005; Hu et al., 2005). Thus, the modulation of hypothalamicAMPK may result in metabolic changes to adapt the organism to newnutritional conditions.

Studies on the metabolic effects of the central administration ofinsulin (Obici et al., 2002), leptin (Obici et al., 2003), and free fattyacids (Morgan et al., 2004) have shown that the metabolic pathwayactivated by these hormones and nutrients in the hypothalamus arealso involved in the control of liver glucose output and insulin action,in addition to food intake control. In fact, central administration ofleptin enhanced insulin sensitivity, systemic glucose utilization(Haque et al., 1999), and inhibited hypothalamic AMPK (Minokoshiet al., 2004; Gao et al., 2007). In our studies, citrate was able tomodulate glucose homeostasis positively, as observed through thetolerance test (GTT) and the hyperinsulinemic–euglycemic clamp.These finding are consistent with the high phosphorylation ofproteins involved in early insulin signaling events and the liverglycogen content. Interestingly, the treatment with citrate ICVincreased AKT phosphorylation in important tissues for insulin-stimulated glucose uptake (skeletal muscle and white adipose),explaining the results obtained in hyperinsulinemic–euglycemicclamp. The magnitude of this increase was similar to that observedin rats treated with ICV ARA-A, a known pharmacological competitiveinhibitor of AMPK. Thus, our data imply that the modulation of theAMPK/ACC pathway by citrate could be an additional glucosehomeostasis control mechanism.

Analysis of blood biochemical parameters showed that thetreatment with citrate did not alter the glucose and insulin levelsper se, discarding the anorexigenic effect of insulin and nutrients onthe hypothalamus (Minokoshi et al., 2004). However, at the momentthat the tissue extraction was performed (30 min after citrateadministration), the serum corticosterone level reached 105±25 ngmL−1, while the basal levels were 205±52 ng mL−1. Previous reports(Tan and Bonen, 1985; Kumar and Leibowitz, 1988) have shown thatcorticosterone may play an important role in the development ofinsulin resistance in skeletal muscle and affect post-receptor events ofthe insulin transduction signal. Therefore, the decrease in thecorticosterone level observed in citrate-treated rats could be an

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additional mechanism to improve insulin signaling. However, thispossibility needs to be better investigated.

A plausible mechanism by which ICV citrate might exert itseffect on insulin sensitivity would be the mediation by AKTupstream events. In fact, conditions that activate AMPK directlyaffect early insulin signaling events by phosphorylating the IRS-1protein on SER789, reducing the insulin capacity of activating PI3-kinase (Eldar-Finkelman and Krebs, 1997; Ravichandran et al.,2001). More recently, Fediuc and colleagues (Fediuc et al., 2006)showed that the presence of AICAR (an AMPK activator) in thesoleus muscle diminishes the insulin-induced AKT phosphorylation.Thus, citrate ICV may inhibit liver AMPK, avoiding its negativeeffect on insulin signaling. In our system, we observed that centraladministration of citrate is responsible for a smaller liver AMPKphosphorylation than that observed in animals receiving saline. Inthis tissue AKT and upstream proteins (IR and IRS) showed higherphosphorylation in the citrate-treated group than in the saline-treated group, indicating a functional relation between them (AMPKinhibition and AKT activation).

Another attractive possibility is the participation of heterotrimericG proteins. This system has been recognized as an important point ofconvergence of signaling from G protein-linked pathways andtyrosine kinase-mediated pathways (Morris and Malbon, 1999). Theexpression of a constitutively active form of G protein, Gαi2, leads toenhanced glucose tolerance (Chen et al., 1997) and GLUT4 localizationat the plasma membrane in the absence of insulin (Song et al., 2001).AMPK α2 gene knockout mice (AMPKα2−/−) exhibited a significantincrease in daily urinary catecholamine excretion and insulin-stimulated whole-body glucose utilization, suggesting high sympa-thetic activation in these mice (Viollet et al., 2003). In addition, whenGαi2 is overexpressed in vivo, enhanced insulin signaling is observed,probably via the suppression of protein tyrosine phosphatase 1B (Taoet al., 2001). In our model, we observed that GLUT4 at the plasmamembrane was higher in the citrate group than in the control group.Thus, we can speculate that central administration of citrate mightactivate Gαi2 in the epididymal fat pad and in skeletal muscle andimprove insulin signaling. It is important to point out that previous iptreatment with a β3-antagonist prevented the effect of citrate on liverAMPK dephosphorylation and inhibited the previously improved AKTphosphorylation stimulated by insulin. A similar effect was obtainedby Haque et al. (1999) in rats treatedwith propranolol and leptin (ICV).This indicates, at least in part, that improvement in the glucosehomeostasis probably involves an increase in the sympathetic outflowto the peripheral tissues.

These results suggest that in addition to insulin, leptin, andglucose, the presence of an intermediate metabolite in thehypothalamus may promote central and peripheral adjustments inthe biochemical mechanisms related to energy intake and glucosehomeostasis, at least in part due to adrenergic-dependent activation.Thus, central citrate and peripheral insulin have a synergistic role inaugmenting tissue glucose uptake. On the other hand, the impair-ment of the metabolic signals involved in the citrate, leptin, orinsulin pathway in the hypothalamus could be related to impairedglucose homeostasis. How adrenergic system would lead to anincrease in the insulin signal and glucose uptake is now underinvestigation. The knowledge of this mechanism would allow thedevelopment of new drugs for the treatment of insulin resistanceand obesity.

Acknowledgements

This work was supported by grants from Fundação de Amparo aPesquisa do Estado de São Paulo-FAPESP and CNPq. We thank Mr.Luiz Janeri, Mr. Józimo Ferreria, and Mr. Márcio Alves da Cruz fortheir technical assistance and Mr. Laerte J. Silva for the Englishlanguage editing.

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