| HOME | HELP | CONTACT US | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Departamento de Bioquímica, IB, Universidade Estadual de Campinas, Campinas, SP, Brazil1 Departamento de Clinica Médica, Universidade Estadual de Campinas, CEP13.083-970 Campinas, SP, Brazil2 Universidade Braz Cubas, Área da Saúde-Campus I, Av. Francisco Rodrigues Filho, 1233, Mogilar, Mogi das Cruzes CEP 08773-380, SP, Brazil
(Correspondence should be addressed to M A Torsoni; Email: torsoni{at}yahoo.com)
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionDeclaration of InterestReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionDeclaration of InterestReferences |
|---|
AMP-activated protein kinase (AMPK) is an enzyme that senses the cell energy status and regulates fuel availability. It is expressed throughout several brain areas that control food intake and neuroendocrine functions. Physiologically, AMPK is modulated by the AMP:ATP ratio and upstream kinases, tumor suppressor kinase (LKB1) and calcium/calmodulin-dependent protein (CAMKK; Hawley et al. 2003). AMPK integrates hormonal and nutrient signals (Han et al. 2005) in order to regulate food intake and energy homeostasis through effects in the hypothalamus and peripheral tissues (Minokoshi et al. 2004, Kahn et al. 2005, Xue & Kahn 2006). AMPK phosphorylates a number of proteins, including acetyl-CoA carboxylase (ACC), and causes their inactivation and a decrease in the concentration of malonyl-CoA (Carling et al. 1987). Some conditions leading to AMPK activation in the hypothalamus also lead to phosphorylation and inactivation of ACC (Kim et al. 2004). Physiologically, the short-term control of ACC occurs largely through phosphorylation/inhibition by 5'-AMPK and by feed-forward allosteric activation by citrate produced by ATP-citrate lyase (Loftus et al. 2000). Then, the presence of citrate activates ACC and promotes cellular accumulation of malonyl-CoA.
Citrate occupies a pivotal position in cellular metabolism. It is not only an intermediate in the citric acid cycle but also a source of cytosolic acetyl-CoA for the synthesis of fatty acids, isoprenoids, and cholesterol. Recently, a novel sodium-coupled citrate transporter (NaCT) expressed in most brain neurons has been described in mouse (Inoue et al. 2002). The transport of citrate into neuron allows it to participate in the generation of energy and the control of the ACC activity.
As cited above, the citrate produced by the mitochondrial metabolism has an important allosteric effect on ACC. The increase in cellular concentration of citrate occurs in parallel to the increase in nutrient availability. In the cytosolic metabolism, citrate is a known negative modulator of phosphofructokinase, a key enzyme of the glycolysis pathway. Thus, there is a link between the glycolytic pathway and the production of malonyl-CoA. Fluctuations in hypothalamic concentrations of malonyl-CoA during fasting and feeding cycles are caused by changes in the phosphorylation and the activity of ACC (Schwartz et al. 2000). In addition, even relatively small increases in glucose levels after refeeding may inhibit hypothalamic AMPK activity (Minokoshi et al. 2004), and consequently activate ACC.
We hypothesized that citrate might be a physiological modulator of feeding behavior and energy homeostasis through its effect on the activation of ACC and the formation of malonyl-CoA. To test this hypothesis, citrate was injected directly into the lateral ventricle for the evaluation of the phosphorylation of hypothalamic AMPK and ACC and the expression of neuropeptides. These effects were investigated in parallel to feeding behavior, body weight, glucose uptake, and insulin signaling in skeletal muscle and epididymal fat pad. Our results show that intracerebroventricular (ICV) injection of citrate diminishes the phosphorylation of hypothalamic ACC and AMPK. This effect is accompanied by reductions in food intake and body weight. Moreover, enhanced glucose uptake and improved insulin pathway activation in epididymal fat pad and skeletal muscle have been observed.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionDeclaration of InterestReferences |
|---|
Male Wistar–Hannover rats (12 weeks old, 250–280 g) from Universidade Estadual de Campinas animal breeding center were used in all experiments. The rats were maintained at room temperature (25 °C) and in 12 h light:12 h darkness cycles with free access to water and chow, unless indicated otherwise. The rats were chronically instrumented with an ICV cannula and kept under controlled temperature and light–darkness conditions (0700–1900 h) in individual metabolic cages. Seven days after the ICV cannula installation, the rats were tested for cannula function and position and thereafter randomly selected for one of the experimental groups. The general guidelines established by the Brazilian College of Animal Experimentation were followed throughout the study. Briefly, the animals were anesthetized with 50 mg/kg ketamine and 5 mg/kg diazepam (i.p.) and positioned onto a Stoelting stereotaxic apparatus after the loss of cornea and foot reflexes. A stainless steel 23 gauge guide cannula with indwelling 30 gauge obturator was stereotaxically implanted into the lateral cerebral ventricle at pre-established coordinates, 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 and correctly positioned by dipsogenic response elicited after injection of angiotensin II (2 µl of 10–6 M solution; McKinley et al. 2001). All procedures were approved by the Ethical Committee of the Universidade Braz Cubas, Mogi das Cruzes.
Biochemical and hormonal measurements
Plasma insulin was determined by RIA according to a previously described method (Scott et al. 1981). Serum glucose was determined by the glucose oxidase method (Trinder et al. 1969) and plasma corticosterone was determined with a commercially available kit following the manufacturer's (DSL Inc., Webster, TX, USA) instructions. All blood samples were collected from the tail vein.
Determination of hypothalamic malonyl-CoA
The tissue fragments were homogenized after the addition of trichloroacetic acid. Precipitated proteins were removed from the trichloroacetic acid extract by centrifugation and the supernatant was neutralized by extracting the acid five times with ether. Samples were lyophilized and stored at –70 °C until assayed (Corkey et al. 1981, 1988). Acid-soluble acyl-CoA compounds were determined by a modification of the reversed-phase HPLC method of Corkey et al. (1981, 1988). This involved the use of acetonitrile rather than methanol as the organic component of the mobile phase, a flow rate of 0.4 mI/min, and a shallow gradient from buffer to acetonitrile to separate malonyl-CoA from early eluting nucleotides. Malonyl-CoA standard was purchased from Sigma–Aldrich Corp.
Protocols for citrate, adenine 9-β-D-arabinofuranoside (ARA-A), and insulin treatments
Citrate and ARA-A (AMP analog adenine 9-β-D-arabinofuranoside) were administered through ICV injection in all the experiment. For the evaluation of the molecular events of the insulin signal transduction pathway in the epididymal fat pad and skeletal muscle, 0.2 ml saline (0.9% NaCl), either with or without insulin (10–4 M), was injected through the cava vein at 0800 h. In this protocol, the treatment with either ARA-A (2 nmol) or citrate (2.0 µl of 10–2 M) occurred 30 min before insulin injection through the cava vein in fasted rats (food was withdrawn 10 h before the treatment). Citrate, ARA-A, and saline solution were buffered with 153 mM NaCl, 10–1 M sodium–phosphate buffer (pH 7.4) to guarantee the efficiency of the NaCT-mediated citrate uptake.
Intraperitoneal glucose tolerance test (i.p.GTT)
The i.p. GTT was performed after overnight fasting. The rats were anesthetized as described above. After collection of an unchallenged sample (time 0), a solution of 20% glucose (2.0 g/kg body weight) was administered into the peritoneal cavity 30 min after ICV administration of citrate (20 nmol). Tail blood samples were collected at 5, 15, 30, 60, 90, and 120 min for the determination of glucose and insulin concentrations.
Hyperinsulinemic–euglycemic clamp procedures
After 5-h fasting, the animals were anesthetized with 50 mg/kg ketamine and 5 mg/kg diazepam injection (i.p.), and catheters were placed into the left jugular vein (for tracer infusions) and the carotid artery (for blood sampling), as previously described (Prada et al. 2000). Each animal was monitored for food intake and weight gain for 5 days after surgery to ensure complete recovery. Food was removed 10 h before the beginning of the in vivo studies. A 120-min hyperinsulinemic–euglycemic clamp procedure was conducted in catheterized rats, as described previously (Combs et al. 2001), with prime continuous infusion of insulin at a rate of 3.6 mU/kg body weight per minute to raise plasma insulin concentration to 
800–900 pmol/l. Blood samples (20 µl) were collected at 5-min intervals for the immediate measurement of plasma glucose concentration, and 10% unlabeled glucose was infused at variable rates to maintain plasma glucose at fasting levels. Insulin-stimulated whole-body glucose flux was estimated using prime continuous infusion of HPLC-purified [3-3H]glucose (10 µCi bolus, 0.1 µCi/min) throughout the clamp procedure (Rossetti et al. 1997). Blood samples (10 µl) were collected before the start and at the end of the clamp procedure for measurement of plasma insulin concentrations. All infusions were performed using Harvard infusion pumps. At the end of the clamp procedure, the animals were killed by a sodium pentobarbital i.v. injection. The basal glucose turnover rates were measured in separate experiments by continuous infusion of [3-3H]glucose (0.02 µCi/min) for 120 min, and the blood samples (20 µl) were taken at 100, 110, and 120 min after the start of the experiment to determine plasma [3-3H]glucose concentration.
Analytical procedures of hyperinsulinemic–euglycemic clamp
Plasma glucose was measured using a glucometer (Advantage, Roche Molecular Biochemicals). Plasma tracer samples were deproteinized with equal volumes of barium hydroxide and zinc sulfate (0.015 M) and stored overnight at 4 °C. The radioactivity of 3-[3H]glucose in plasma was measured in Ba(OH)2/ZnSO4 precipitate supernatants after evaporation to dryness for the removal of tritiated water. Rates of whole-body glucose uptake and basal glucose turnover were determined as the ratio between the [3-3H]glucose infusion rate (disintegrations per minute) and the specific activity of plasma glucose (disintegrations per minute per milligram glucose) during the final 30 min of the respective experiments under steady-state conditions. Glucose transport activity in skeletal muscle and fat was calculated from plasma 2-[14C]DG profile, as described previously (Kraegen et al. 1958, Ferre et al. 1985).
Food intake and body weight measurements
To measure individual food intake, the animals were divided into two groups: i) feeding rats, animals given free access to standard rodent chow and ii) fasted rats, animals fasted during light phase (8 h). Food intake was evaluated during the dark phase after ICV administration of either saline or citrate (20 nmol) at 1800 h.
In another experimental protocol, the rats were maintained in individual cages with free access to standard rodent chow and water for 2 days for adaptation. Later, the animals received ICV administration of either citrate or saline every day at 1800 h. Food intake measurements were performed in the next 12 h for 7 experimental days. To differentiate the effects of citrate administration per se versus effects induced by food intake inhibition, a citrate pair-fed group was included. Body weight, epididymal fat pad, and perirenal fat were obtained from control, citrate-treated rats, and the citrate pair-fed group after 9 days (2 days for adaptation plus 7 experimental days). Citrate was administered immediately before the beginning of the nocturnal cycle.
RNA preparation for reverse transcription-PCR
Total hypothalamic and hepatic RNA were extracted using Trizol (Life Technologies) reagent according to the manufacturer's guidelines. Total RNA was rendered genomic DNA-free by digestion with RNase-free DNase (RQ1; Promega).
Semi-quantitative reverse transcription-PCR
Seven micrograms of total RNA were reverse transcribed with SuperScript reverse transcriptase (200 U/µl) using oligo (dT) (50 mmol/l) in 30 µl reaction volume (5 x RT buffer, 10 mmol/l dNTP, and 40 U/µl RNase-free inhibitor). The reverse transcriptions involved 50-min incubation at 42 °C and 15-min incubation at 70 °C. PCR products were submitted to 1.5% agarose gel electrophoresis containing ethidium bromide and visualized under u.v. light excitation. Photo-documentation was performed using the Nucleovision System (NucleoTech, San Mateo, CA, USA) and band quantification was performed using Gel Expert Software (NucleoTech). RPS-29 (ribosomal protein S29) of all samples was amplified and used as internal qualitative and quantitative controls. The semi-quantitative expression (SE) of genes of interest was calculated using the formula: SE=pixel area of product/pixel area of RPS-29x100. The primers used and the PCR conditions were: RPS-29 (NCBI: NM012876), sense: 5'-AGG CAA GAT GGG TCA CCA GC-3', antisense: 5'-AGT CGA ATC ATC CAT TCA GGT CG-3' (fragment: 202 bp; Tm: 57 °C; amplification: 27 cycles); POMC (proopiomelanocortin; NCBI: AF510391), sense: 5'-CTC CTG CTT CAG ACC TCC AT-3', antisense: 5'-TTG GGG TAC ACC TTC ACA GG-3' (fragment: 398 bp; Tm: 63 °C; amplification: 32 cycles); NPY (neuropeptide Y; NCBI: NM012614), sense: 5'-AGA GAT CCA GCC CTG AGA CA-3', antisense: 5'-AAC GAC AAC AAG GGA AAT GG-3' (fragment: 236 bp; Tm: 62 °C; amplification: 31 cycles); CRH (corticotropin-releasing hormone; NCBI: NM031019), sense: 5'-ATC CGC ATG GGT GAA GAA TA-3', antisense: 5'-AAG CGC AAC ATT TCA TTT CC-3' (fragment: 408 pb; Tm: 62 °C; amplification: 31 cycles).
Tissue extraction, immunoprecipitation, and immunoblotting
The rats were anesthetized after specific treatments and tissues samples were obtained and homogenized in freshly prepared ice-cold buffer (1% Triton X-100, 100 mM TRIS, pH 7.4, 100 mM sodium pyrophosphate, 100 mM sodium fluoride, 10 mM EDTA, 10 mM sodium vanadate, 2 mM phenylmethylsulphonyl fluoride (PMSF), and 0.01 mg aprotinin/ml). The insoluble material was removed by centrifugation (10 000 g) for 25 min at 4 °C. Aliquots of the resulting supernatants containing 2 mg total protein were used for immunoprecipitation with either specific antibodies (anti-IR (rabbit; sc-711), or anti-IR substrate-1 (IRS1; rabbit; sc-559), or anti-IRS2 (goat; sc-1555)), all from Santa Cruz Biotechnology (Santa Cruz, CA, USA) at 4 °C overnight, followed by the addition of protein A–Sepharose 6 MB (Pharmacia) for 2 h and centrifuged at 10 000 g for 15 min at 4 °C. The pellets were washed three times in ice-cold buffer (0.5% Triton X-100, 100 mM Tris, pH 7.4, 10 mM EDTA, and 2 mM sodium vanadate), resuspended in Laemmli sample buffer, and boiled for 5 min before separation in SDS-PAGE using a miniature slab gel apparatus (Bio-Rad). Electrotransfer of proteins from the gel to nitrocellulose was performed for 90 min at 120 V (constant). The nitrocellulose transfers were probed with specific antibodies. The blots were subsequently incubated with 125I-labeled protein A (Amersham). For direct immunoblot analysis, 0.2 mg protein from tissue extracts was separated by SDS-PAGE, transferred to nitrocellulose membranes, and blotted with specific antibodies, anti-phospho-AMPK, and anti-phospho-ACC (Cell Signaling Technology, Danvers, MA, USA). Subsequently, the blots were incubated with 125I-labeled protein A (Amersham). The results were visualized by autoradiography with pre-flashed Kodak XAR film. Band intensities were quantified by optical densitometry of developed autoradiographs (Scion Image software; ScionCorp, Frederick, MD, USA).
Data presentation and statistical analysis
All numerical results are expressed as means±S.E.M. of the indicated number of experiments. Blot results are presented as direct band comparisons in autoradiographs and quantified by densitometry using the Scion Image software (ScionCorp). Student's t-tests of unpaired samples and ANOVA for multiple comparisons were used as appropriate. Post hoc test (Tukey) was employed when required. The level of significance was set at P<0.05.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionDeclaration of InterestReferences |
|---|
An acute ICV injection of 20 nmol of citrate in fed animals elicited a decrease (35%) in food intake (Fig. 1A). A similar effect was observed in animals ICV injected a competitive inhibitor of AMPK (ARA-A). In fasted animals, the citrate effect was similar to that observed in fed animals (Fig. 1B). Food intake was evaluated at early time points (10 and 30 min after the exogenous administration of citrate), but the food intake measured value in this period was very small. Since the AMPK/ACC pathway plays an important role in the feeding behavior, we evaluated the phosphorylation of hypothalamic AMPK and ACC 30 min after the ICV administration of citrate to fed (Fig. 1C and D) and fasted rats at different times after the exogenous administration of citrate (Fig. 1E). AMPK phosphorylation was reduced significantly in both groups (fed and fasted animals) after ICV administration of citrate (54 and 62% respectively) comparatively with values in animals ICV-administered saline. The evaluation of the hypothalamic phosphorylation of AMPK at 5, 10, and 15 min after the ICV administration of citrate resulted in an effect on the level of phosphorylation of AMPK similar to that observed at 30 min (Fig. 1E). Similarly to citrate, ICV administration of a competitive AMPK inhibitor (ARA-A) also reduced AMPK phosphorylation and ACC phosphorylation in the hypothalamus (Fig. 1C and D). Interestingly, hypothalamic phosphorylation of ACC was reduced when either citrate or ARA-A (71 and 52% respectively) was administered to fed rats, showing that citrate can reduce the kinase activity of hypothalamic AMPK. To evaluate the potential mechanism involved in the effects on the food intake, we quantified the level of malonyl-CoA in the hypothalamic extract. Consistent with its effect on AMPK/ACC, exogenous administration of citrate increased malonyl-CoA in the hypothalamus when compared with the results of animals that received saline (7.4±1.0 vs 5.2±0.9 pmol/mg protein respectively).
|
|
Food intake was evaluated after single daily ICV injection of either citrate or saline vehicle (control) just before the darkness cycle onset (1800 h) for 7 days. Food intake was 25% lower in the citrate ICV group, whereas no significant change in food intake was observed in the saline ICV group during the experimental period (7 days; Fig. 3A). At the end of the experimental protocol, the cumulative body weight was positive in the saline group (+35 g), whereas a negative body weight variation was detected in animals treated with citrate (5 g). Interestingly, the citrate pair-fed group showed a positive body weight variation (+23 g) significantly different from that of the citrate group (Fig. 3B). Concordantly, the epididymal fat pad (–1.7 g) and perirenal fat (1.4 g) were significantly reduced after 7 days of treatment with citrate. However, in the citrate pair-fed group only the epididymal fat pad was significantly reduced (Fig. 3C and D) when compared with the control group result.
|
|
|
|
|
As expected, insulin infusion through the cava vein (closed bars) promoted an increase in tyrosine phosphorylation (IR, IRS1, and IRS2) and serine phosphorylation (AKT) in skeletal muscle (Fig. 7A–D) when compared with the results of animals treated with saline (open bars). However, insulinstimulated phosphorylation of IR, IRS1, IRS2, and AKT was powered in animals previously treated with either citrate (citrate ICV) or ARA-A (ARA-A ICV) ICV injection.
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionDeclaration of InterestReferences |
|---|
The anorexigenic effect of citrate is corroborated by data obtained after evaluation of hypothalamic NPY, POMC, and CRH mRNA levels. In our study, citrate could also promote the reduction of NPY mRNA expression in the hypothalamus. Additionally, POMC and CRH expressions were increased in fasted rats after citrate ICV treatment. In another study, AMPK inhibition in the hypothalamus also was sufficient to decrease food intake and suppress mRNA expression of NPY (Minokoshi et al. 2004). Additionally, the plasma corticosterone level in fasted rats (basal level) was markedly diminished (42%) by citrate ICV injection. Other authors showed that the blood corticosterone level presented a prandial fall subsequent to feeding (Honma et al. 1986, Jahng et al. 2005). Therefore, we can admit that citrate ICV administration changed the feeding behavior and the molecular characteristic of the hypothalamus despite the effect of fasting in rats.
In addition to the effects on food intake and neuropeptide expression in the hypothalamus, hypothalamic AMPK seems to play an important role in the central control of the peripheral metabolism. Hypothalamic AMPK modulation mediates glycogen synthesis in muscle (Perrin et al. 2004) and fatty acid oxidation in muscle is induced by early activation of AMPK by leptin acting directly on muscle, whereas later activation depends on leptin functioning through the hypothalamic–sympathetic nervous system axis (Minokoshi et al. 2002). Furthermore, AMPK knockout mice presented impaired glucose homeostasis (Viollet et al. 2003). Although the mechanisms are not completely known yet, these studies established an important correlation between AMPK inhibition and glucose homeostasis. In accordance to this possibility, our results showed that rats treated with citrate by ICV infusion display improved glucose homeostasis as evaluated by GTT and euglycemic–hyperinsulinemic clamp. The possibility of citrate itself inducing insulin secretion was investigated, but it was not confirmed, since the blood insulin level did not change after citrate ICV administration (up to 60 min) in fasted rats. Therefore, we suspected that an improvement in glucose homeostasis could be caused by a more efficient stimulus of the elements of the insulin signaling pathway.
Finally, with regard to the citrate ICV effect on protein phosphorylation stimulated by insulin, we evaluated the phosphorylation level of IR, IRS1, IRS2, and AKT. Interestingly, the citrate treatment (ICV) promoted an improvement in the insulin-stimulated phosphorylation of IR, IRS1, and AKT proteins in the epididymal fat pad. On the other hand, in skeletal muscle, IR, IRS2, and AKT proteins presented an increase in insulin-stimulated phosphorylation after citrate administration. These effects were also obtained when the AMP analog, ARA-A, an AMPK competitive inhibitor, was ICV injected. Moreover, skeletal muscle of rats treated with citrate for 7 days presented a glycogen level (59%) higher the control group (data not shown) did. Since citrate improved insulin signaling, a more robust control of the glycogen synthesis activation and inhibition of glycogen breakdown seem to have occurred during the experimental protocol (7 days). Thus, the inhibition of hypothalamic AMPK seems to have particular importance for events observed in peripheral tissues. In contrast to our findings, studies revealed that total 
2AMPK knockout mice display insulin resistance, impaired AICAR and glucose tolerance, impaired glucose-stimulated insulin secretion, reduced insulin-stimulated whole-body glucose utilization and skeletal muscle glycogen synthesis, and elevated catecholamine excretion in urine (Viollet et al. 2003). Nevertheless, in our study, only hypothalamic AMPK was inhibited by treatment. Thus, we believe that the peripheral tissue effects are secondary to the inhibition of hypothalamic AMPK. We cannot disregard the involvement of hypothalamic ACC in these effects, particularly because ACC is responsible for the synthesis of malonyl-CoA, which has been attributed an important role in the energy homeostasis (Wolfgang & Lane 2006). However, the mechanisms of such effects are unclear, but activation of the hypothalamic–sympathetic nervous system is likely.
Although several possibilities may be discussed to try to elucidate the mechanisms involved in the inactivation of hypothalamic AMPK and its effects on glucose homeostasis, a new component that may enhance the insulin signaling pathway protein should be considered. Heterotrimeric G-proteins have been recognized as important points of convergence of signaling from G-protein-linked pathways and tyrosine kinase-mediated pathways (Morris & Malbon 1999). The expression of a constitutively active form of G-protein, Gi2, leads to enhanced glucose tolerance (Chen et al. 1997) and glucose transporter type 4 (GLUT4) localization at the plasma membrane in the absence of insulin (Song et al. 2001). In addition, when Gi2 is overexpressed in vivo, enhanced insulin signaling is observed, probably via suppression of protein, tyrosine phosphatase 1B (Tao et al. 2001). Thus, we can speculate that central administration of citrate might activate Gi2 in the epididymal fat pad and in skeletal muscle and improve insulin signaling.
The biochemical events related to the presence of citrate in the cytoplasm are well known. Citrate is an important negative modulator of phosphofructokinase (PFK) and would decrease the glycolytic flux and the ATP level, leading to AMPK activation. Thus, the exogenous administration of citrate would be expected to activate AMPK. Later, citrate would be transferred to the mitochondria, and thus the activation of AMPK would be reduced. However, AMPK is regulated by fasting and feeding in such a way that during fasting, AMPK is active and imposes a negative regulation on ACC. By contrast, after feeding, AMPK is rapidly inactivated and ACC activity is restored, as previously demonstrated by our group (Roman et al. 2005). The data shown in the present paper were obtained from fasted animals that presented increased phosphorylation of hypothalamic AMPK. The reduced phosphorylation of hypothalamic AMPK for all the times after exogenous citrate administration investigated suggests that, like glucose, citrate could elevate the ATP level in the cytoplasm very fast. Consistent with the important effect attributed to citrate administered exogenously, food intake stimulated by fasting was diminished by citrate ICV injection in fasted animals.
In conclusion, the increase in the concentration of cytoplasmic citrate in neurons may indicate the nutrient availability to the central nervous system and modulate glucose homeostasis and energy intake. A point of note, the signal is strong enough to change feeding behavior and glucose homeostasis even in fasted animals. Taken together, these results provide a new target for the study of the central regulatory mechanism of body energy disposal. In addition to enzymes AMPK and ACC, the enzymes involved in the citrate metabolism are potential targets of studies of therapeutic agents for diabetes control and body weight reduction.
|
Declaration of Interest |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionDeclaration of InterestReferences |
|---|
|
Funding |
|---|
|
Acknowledgements |
|---|
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionDeclaration of InterestReferences |
|---|
Carling D, Zammit V & Hardie DG 1987 A common bicyclic protein kinase cascade inactivates the regulatory enzymes of fatty acid and cholesterol biosynthesis. FEBS Letters 223 217–222.[CrossRef][Web of Science][Medline]
Chen JF, Guo JH, Moxham CM, Wang HY & Malbon CC 1997 Conditional, tissue-specific expression of Q205L G alpha i2 in vivo mimics insulin action. Journal of Molecular Medicine 75 283–289.[CrossRef][Web of Science][Medline]
Combs TP, Berg AH, Obici S, Scherer PE & Rossetti L 2001 Endogenous glucose production is inhibited by the adipose-derived protein Acrp30. Journal of Clinical Investigation 108 1875–1881.[CrossRef][Web of Science][Medline]
Corkey BE, Brandt M, Williams RJ & Williamson JR 1981 Assay of short-chain acyl coenzyme A intermediates in tissue extracts by high-pressure liquid chromatography. Analytical Biochemistry 118 30–41.[CrossRef][Web of Science][Medline]
Corkey BE, Hale DE, Glennon MC, Kelley RI, Coates PM, Kilpatrick L & Stanley CA 1988 Relationship between unusual hepatic acyl coenzyme A profiles and the pathogenesis of Reye syndrome. Journal of Clinical Investigation 82 782–788.[Web of Science][Medline]
Elmquist JK 2001 Hypothalamic pathways underlying the endocrine, autonomic, and behavioral effects of leptin. Physiology and Behavior 74 703–708.[CrossRef][Medline]
Ferre P, Leturque A, Burnol AF, Penicaud L & Girard L 1985 A method to quantify glucose utilization in vivo in the anaesthetized rat. Biochemical Journal 228 103–110.[Web of Science][Medline]
Friedman JM & Halaas JL 1998 Leptin and the regulation of body weight in mammals. Nature 395 763–770.[CrossRef][Medline]
Gao S, Kinzig KP, Aja S, Scott KA, Keung W, Kelly S, Strynadka K, Chohnan S, Smith WW, Tamashiro KL et al. 2007 Leptin activates hypothalamic acetyl-CoA carboxylase to inhibit food intake. PNAS 104 17358–17363.
Han SM, Namkoong C, Jang PG, Park IS, Hong SW, Katakami H, Chun S, Kim SW, Park JY, Lee KU et al. 2005 Hypothalamic AMP-activated protein kinase mediates counter-regulatory responses to hypoglycaemia in rats. Diabetologia 48 2170–2178.[CrossRef][Web of Science][Medline]
Hawley SA, Boudeau J, Reid JL, Mustard KJ, Udd L, Makela TP, Alessi DR & Hardie DG 2003 Complexes between the LKB1 tumor suppressor, STRAD /β and MO25 /β are upstream kinases in the AMP-activated protein kinase cascade. Journal of Biology 2 28–34.[CrossRef][Medline]
Honma K, Honma S, Hirai T, Katsuno Y & Hiroshige T 1986 Food ingestion is more important to plasma corticosterone dynamics than water intake in rats under restricted daily feeding. Physiology and Behavior 37 791–795.[CrossRef][Medline]
Inoue K, Zhuang L, Maddox DM, Smith SB & Ganapathy V 2002 Structure, function, and expression pattern of a novel sodium-coupled citrate transporter (NaCT) cloned from mammalian brain. Journal of Biological Chemistry 277 39469–39476.
Jahng JW, Lee JY, Yoo SB, Kim Y, Ryu V, Kang DW & Lee JH 2005 Refeeding-induced expression of neuronal nitric oxide synthase in the rat paraventricular nucleus. Brain Research 1048 185–192.[CrossRef][Web of Science][Medline]
Kahn BB, Alquier T, Carling D & Hardie DG 2005 AMP-activated protein kinase: ancient energy gauge provides clues to modern understanding of metabolism. Cell Metabolism 1 15–25.[CrossRef][Web of Science][Medline]
Kalra SP, Dube MG, Sahu A, Phelps CP & Kalra PS 1991 Neuropeptide Y secretion increases in the paraventricular nucleus in association with increased appetite for food. PNAS 88 10931–10935.
Kim EK, Miller I, Aja S, Landree LE, Pinn M, McFadden J, Kuhajda FP, Moran TH & Ronnett GV 2004 Anti-obesity effects of alpha-lipoic acid mediated by suppression of hypothalamic AMP-activated protein kinase. Journal of Biological Chemistry 279 19970–19976.
Kraegen EW, James DE, Jenkins AB & Chisholm DJ 1958 Dose–response curves for in vivo insulin sensitivity in individual tissue in rats. American Journal of Physiology 248 E353–E362.
Kubota N, Yano W, Kubota T, Yamauchi T, Itoh S, Kumagai H, Kozono H, Takamoto I, Okamoto S, Shiuchi T et al. 2007 Adiponectin stimulates AMP-activated protein kinase in the hypothalamus and increases food intake. Cell Metabolism 6 55–68.[CrossRef][Web of Science][Medline]
Lane MD, Hu Z, Cha SH, Dai Y, Wolfgang M & Sidhaye A 2005 Role of malonyl-CoA in the hypothalamic control of food intake and energy expenditure. Biochemical Society Transactions 33 1063–1067.[CrossRef][Web of Science][Medline]
Loftus TM, Jaworsky DE, Frehywot GL, Townsend CA, Ronnett GV, Lane MD & Kuhajda FP 2000 Reduced food intake and body weight in mice treated with fatty acid synthase inhibitors. Science 288 2379–2381.
McKinley MJ, Allen AM, May CN, McAllen RM, Oldfield BJ, Sly D & Mendelsohn FA 2001 Neural pathways from the lamina terminalis influencing cardiovascular and body fluid homeostasis. Clinical and Experimental Pharmacology and Physiology 28 990–992.[CrossRef][Web of Science][Medline]
Michelotto JB, Carvalheira JB, Saad MJ & Gontijo JA 2002 Effects of intraceroventricular insulin microinjection on renal sodium handandling in kidney-denervated rats. Brain Research Bulletin 57 613–618.[CrossRef][Web of Science][Medline]
Minokoshi Y, Kim YB, Peroni OD, Fryer LG, Muller C, Carling D & Kahn BB 2002 Leptin stimulates fatty acid fatty acid oxidation by activating AMP-activated protein kinase. Nature 415 339–343.[CrossRef][Medline]
Minokoshi Y, Alquier T, Furukawa N, Kim YB, Lee A, Xue B, Mu J, Foufelle F, Ferre P, Birnbaum MJ et al. 2004 AMP-kinase regulates food intake by responding to hormonal and nutrient signals in the hypothalamus. Nature 428 569–574.[CrossRef][Medline]
Morris A & Malbon CC 1999 Physiological regulation of G protein-linked signaling. Physiological Reviews 79 1373–1430.
Perrin C, Knauf C & Burcelin R 2004 Intracerebroventricular infusion of glucose, insulin, and the adenosine monophosphate-activated kinase activator, 5-aminoimidazole-4-carboxamide-1-β-dribofuranoside, controls muscle glycogen synthesis. Endocrinology 145 4025–4033.
Prada PO, Okamoto MM, Furukawa LN, Machado UF, Heimann J & Dolnikoff MS 2000 High- or low-salt diet from weaning to adulthood: effect on insulin sensitivity in Wistar rats. Hypertension 35 424–429.
Roman EA, Cesquini M, Stoppa GR, Carvalheira JB, Torsoni MA & Velloso LA 2005 Activation of AMPK in rat hypothalamus participates in cold-induced resistance to nutrient-dependent anorexigenic signals. Journal of Physiology 568 993–1001.
Rossetti L, Massilon D, Barzilar N, Vuguin P, Chen W, Hawkins M, Wu J & Wang J 1997 Short term effects of leptin on hepatic gluconeogenesis and in vivo insulin action. Journal of Biological Chemistry 272 277758–277763.
Schwartz MW, Woods SC, Porte D Jr, Seeley RJ & Baskin DG 2000 Central nervous system control of food intake. Nature 404 661–671.[Medline]
Scott AM, Atwater I & Rojas E 1981 A method for the simultaneous measurement of insulin release and B cell membrane potential in single mouse islets of Langerhans. Diabetologia 21 470–475.[Web of Science][Medline]
Song X, Zheng X, Malbon CC & Wang H 2001 G i2 enhances in vivo activation of and insulin signaling to GLUT4. Journal of Biological Chemistry 276 34651–64658.
Tao J, Malbon CC & Wang HY 2001 Galpha(i2) enhances insulin signaling via suppression of protein-tyrosine phosphatase 1B. Journal of Biological Chemistry 276 39705–39712.
Trinder N, Woodhouse CD & Renton CP 1969 The effect of vitamin E and selenium on the incidence of retained placentae in dairy cows. Veterinary Record 85 550–553.[Web of Science][Medline]
Viollet B, Andreelli AF, Jorgensen SB, Perrin C, Flamez D, Mu J, Wojtaszewski JF, Schuit FC, Birnbaum M, Richter E et al. 2003 The AMP-activated protein kinase 2 catalytic subunit controls whole-body insulin sensitivity. Journal of Clinical Investigation 111 91–98.[CrossRef][Web of Science][Medline]
Wolfgang MJ & Lane MD 2006 The role of hypothalamic malonyl-CoA in energy homeostasis. Journal of Biological Chemistry 281 37265–37269.
Xue B & Kahn BB 2006 AMPK integrates nutrient and hormonal signals to regulate food intake and energy balance through effects in the hypothalamus and peripheral tissues. Journal of Physiology 574 73–83.
Received in final form 5 May 2008
Accepted 8 May 2008
Made available online as an Accepted Preprint 9 May 2008
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | CONTACT US | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
0.05 for citrate ICV versus saline ICV, and ARA-A ICV versus saline ICV.
) or citrate (
) ICV (20 nmol) after overnight fasting and 30 min after the treatment with a solution of 20% glucose (2.0 g/kg body weight) administered into the peritoneal cavity. Tail blood samples were collected for the determination of glucose and insulin concentrations. Data are expressed as mean±S.E.M. of five animals/group. *P
