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Endocrinology, doi:10.1210/en.2006-0223
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Endocrinology Vol. 147, No. 11 5470-5479
Copyright © 2006 by The Endocrine Society

Leptin Inhibits Apoptosis in Thymus through a Janus Kinase-2-Independent, Insulin Receptor Substrate-1/Phosphatidylinositol-3 Kinase-Dependent Pathway

Eli Mansour, Fernanda G. Pereira, Eliana P. Araújo, Maria E. C. Amaral, Joseane Morari, Natasha R. Ferraroni, Diogenes S. Ferreira, Irene Lorand-Metze and Lício A. Velloso

Department of Internal Medicine, State University of Campinas, 13083-970 Campinas SP, Brazil

Address all correspondence and requests for reprints to: Dr. Licio A. Velloso, Departamento de Clínica Médica, Faculdade de Ciências Médicas-State University of Campinas, 13083-970, Campinas SP, Brazil. E-mail: lavelloso{at}fcm.unicamp.br.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The cytokine-like hormone leptin is known to exert important functions on the modulation of immune responses. Some of these effects are dependent on the property of leptin to modulate the apoptosis of thymic cells. In the present study, we used Wistar rats to investigate the molecular mechanisms involved in leptin-dependent control of apoptosis in thymus. Apoptosis was evaluated by flow cytometry and ELISA for nucleosome determination, whereas signal transduction was evaluated by immunoprecipitation, immunoblot, and confocal microscopy. The Ob receptor (ObR) was expressed in most thymic cells and its relative amount reduced progressively during thymocyte maturation. ObR expression was colocalized with Janus kinase (JAK)-2 and signal transducer and activator of transcription-3, and an acute, in vivo, injection of leptin promoted the tyrosine phosphorylation of JAK-2 and the engagement of signal transducer and activator of transcription-3. The treatment with leptin also led to the tyrosine phosphorylation of insulin receptor substrate (IRS)-1 and serine phosphorylation of Akt. Chronic treatment with leptin reduced thymic apoptosis, an effect that was not inhibited by the JAK inhibitor AG490 but was significantly inhibited by the phosphatidylinositol 3-kinase inhibitor LY294002 and an antisense oligonucleotide to IRS-1. Thus, leptin inhibits the apoptosis of thymic cells through a mechanism that is independent of the activation of JAK-2 but depends on the engagement of the IRS-1/phosphatidylinositol 3-kinase pathway.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
LEPTIN, THE PRODUCT of the ob gene, is a cytokine-like hormone, produced mostly by the adipose tissue in direct proportion to whole-body fat mass (1). In recent years a role for leptin in the regulation of immune response has been uncovered (2). Apparently leptin acts as a link between the nutritional status and the control of immune system activity (2). Under physiological conditions, the increase in body fat mass that follows a period of overeating, leads to an increased leptin activity in specialized neurons of the hypothalamus, inducing energy expenditure and reducing food intake (1, 3). In addition, leptin regulates different facets of the immune response, such as the promotion of an enhancement of peripheral T cell activity and proliferation (4, 5), activation of monocyte response (6, 7), regulation of cytokine production (8), and modulation of immune response during autoimmunity (9).

Most leptin actions are delivered through the activation of the IL-6/gp 130-like Ob receptor (ObR) (10). Like other members of the class I cytokine family, the ObR lacks intrinsic tyrosine kinase activity and depends on the activation of an intracellular kinase to achieve full engagement of its intracellular signal transduction pathway (10). Upon leptin binding, the ObR engages Janus kinase (JAK)-2, inducing its autophosphorylation at tyrosine residues, which is followed by tyrosine phosphorylation of the ObR and subsequent recruitment, tyrosine phosphorylation, and induction of dimerization of signal transducer and activator of transcription (STAT)-3, which migrates to the nucleus and finally regulates gene transcription (10, 11, 12). Besides its effects on the classical JAK/STAT signaling pathway, which provides a direct access to the nucleus, leptin activates several other intracellular signaling pathways such as the MAPK cascade (13, 14, 15), phosphatidylinositol 3-kinase (PI 3-kinase)/Akt (16), SH2-B (17) and insulin receptor substrate (IRS)-1 (18). Through these pathways, leptin may be integrated to a complex intracellular cross talk system that regulates functions such as cell growth, mitogenesis, metabolism, and apoptosis (1, 2, 19).

One of the most remarkable aspects of malnutrition is the atrophy of the thymus and the development of immunosuppression (20, 21). Starvation causes a loss of normal thymic architecture and reduces the number of cortical thymocytes by increasing the rate of apoptosis (22). In addition, starvation and chronic malnutrition promotes a fall in leptin levels (23), and this phenomenon has been proposed to play a role in the anomalous thymic morphology and function observed during nutritional deprivation states (22). This hypothesis has been further supported by the fact that both humans and rodents with defective leptin production present a significant restoration of different aspects of the immune response when treated with exogenous leptin (8, 22, 24) and that the treatment of leptin-deficient ob/ob mice with exogenous leptin reduces thymic atrophy by increasing its cellularity (22).

Although the functional effects of leptin on thymic cellularity and apoptosis have been well characterized, the molecular mechanisms involved in this control are poorly understood. Therefore, the objective of the present study was to evaluate the intracellular transduction pathways that participate in leptin-induced inhibition of apoptosis in the thymus of Wistar rats.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Experimental animals
Four-week-old male Wistar rats and Lepdb (db/db) and C57BLKS/J mice were obtained from the University of Campinas Breeding Center. The Lepdb (db/db) mice were originally purchased from the Jackson Laboratory (Bar Harbor, ME) and are currently established as a colony at the University of Campinas Breeding Center. The animals were allowed access to standard rodent chow and water ad libitum. All experiments involving animals were in accordance with the guidelines of the Brazilian College for Animal Experimentation and approved by the University of Campinas Ethical Committee. Room temperature was maintained between 21 and 23 C with 12-h light, 12-h dark cycle. The animals were age matched for individual experiments and randomly distributed into treatment or control groups.

Materials
Antibodies against JAK-2 (sc-278), STAT-3 (sc-483), phosphotyrosine (sc-508), IRS-1 (sc-559) ObR (sc-8325), and phospho-(Ser473) Akt (sc-9271) were from Santa Cruz Biotechnology (Santa Cruz, CA). Conjugated mouse antirat CD3-fluorescein isothiocyanate isomer 1 (FITC), CD4-phycoerythrin (RPE), and CD8-RPE-Cy5 were from Serotec, Ltd. (Oxford, UK). 125I-protein A Sepharose and nitrocellulose paper (Hybond ECL, 0.45 µm) were from Amersham (Buckinghamshire, UK). Protein A Sepharose 6 MB was from Pharmacia (Uppsala, Sweden), and protein A Agarose (AG-Plus) (sc-2003) was from Santa Cruz Biotechnology. Leptin, the JAK inhibitor AG490 (tyrphostin B42), and the PI 3-kinase inhibitor LY294002 were acquired from Calbiochem (La Jolla, CA). Sodium amobarbital was purchased from Eli Lilly & Co. (Indianapolis, IN). Tris base, phenylmethylsulfonylfluoride, aprotinin, dithiothreitol, Triton X-100, Tween 20, glycerol, affinity-purified rabbit antimouse IgG, and BSA (fraction V) were obtained from Sigma (St. Louis, MO). RPMI 1640 and reagents for cell culture were purchased from Invitrogen Corp. (Carlsbad, CA). Sense (5'-ACC CAC TCC TAT CCC G-3') (IRS-1SO) and antisense (5'-CGG GAT AGG AGT GGG T-3') (IRS-1ASO) phosphorthioate oligonucleotides specific for IRS-1 were produced by Invitrogen (25, 26, 27). Sequence was selected among three unrelated pairs of oligonucleotides on the basis of their ability to block IRS-1 protein expression as evaluated by immunoblot of total protein extracts of thymus tissue using specific anti-IRS-1 antibodies. The oligonucleotide sequences were submitted to BLAST analyses (www.ncbi.nlm.nih.gov) and matched only for the Rattus norvegicus IRS-1 coding sequence (NCBI/NM 012969). The nucleosome ELISA kit (catalog no. QIA25) was acquired from Oncogene Research Products (Boston, MA), and the flow cytometry annexin-V apoptosis detection kit (Apoptest K2350) was purchased from Dako Corp. (Carpinteria, CA). The leptin ELISA kit was purchased from Linco Research Inc. (St. Charles, MO). TRIzol reagent was purchased from Invitrogen, and Moloney murine leukemia virus reverse transcriptase was from CLONTECH (Mountain View, CA).

Protocols for acute treatment with leptin and protein analysis by immunoprecipitation and immunoblotting
Rats were anesthetized by ip injection of sodium amobarbital (15 mg/kg body weight) and submitted to the surgical procedure as soon as the anesthesia was assured by the loss of pedal and corneal reflexes. The abdominal cavity was opened, the portal vein was exposed, and in vivo stimulation was obtained by the injection of 400 µl saline (0.9% NaCl), insulin (10–6 M), or leptin (10–6, 10–8, or 10–10 M). After the predetermined elapsed time, the thymus was removed after a thoracotomy. The tissue was minced coarsely and homogenized immediately in extraction buffer [1% Triton X-100 and 100 mM Tris (pH 7.4) containing 100 mM sodium pyrophosphate, 100 mM sodium fluoride, 10 mM EDTA, 10 mM sodium vanadate, 2 mM phenylmethylsulfonylfluoride, and 0.1 mg aprotinin/ml] at 4 C with a Polytron PTA 20S generator (model PT 10/35; Brinkmann Instruments, Inc., Westbury, NY) operated at maximum speed for 30 sec. The extracts were centrifuged at 9000 x g and 4 C in a 70.1 Ti rotor (Beckman, Palo Alto, CA) for 20 min to remove insoluble material, and the supernatants were used for immunoprecipitation with anti-JAK-2, -STAT-3, or -IRS-1 antibodies, and the technical procedures were performed as previously described (28, 29). Protein quantification in the supernatants was determined by the Bradford method (30). Immunoprecipitates were separated by SDS-PAGE, transferred to nitrocellulose membranes, and blotted with antibodies antiphosphotyrosine. In direct immunoblotting experiments, total protein extracts were separated by SDS-PAGE, transferred to nitrocellulose membranes, and antiphospho-(Ser473) Akt and anti-IRS-1 antibodies used for blotting. Visualization of specific protein bands was performed by incubating membranes with 125 I-protein A followed by exposure to x-ray films (Kodak, Rochester, NY). In some experiments, the rats were pretreated with 100 µl AG490 10–4 M or 400 µl LY294002 5.0 µM and then submitted to acute treatment with leptin.

Protocols for chronic treatment with leptin and evaluation of apoptosis
For these experiments, the rats were randomly divided into eight groups and treated by ip injection according to one of the following protocols: 400 µl saline; 400 µl leptin (10–6 M); 100 µl AG 490 (10–4 M) + 400 µl leptin; 400 µl LY294002 (5 µM) + 400 µl leptin; 100 µl IRS-1SO (4.0 nmol) + 400 µl leptin; 100 µl IRS-1SO (4.0 nmol) + 400 µl saline solution; 100 µl IRS-1ASO (4.0 nmol) + 400 µl leptin; or 100 µl IRS-1ASO (4.0 nmol) + 400 µl saline solution. The treatment consisted of two doses each day for 3 consecutive days. On the morning of the fourth day, the animals were anesthetized as described earlier, and the thymus was removed by thoracotomy. Suspensions of thymocytes in PBS/2% fetal calf serum (Cult Lab, Campinas, Brazil) were obtained using a Potter glass and used for apoptosis detection by flow cytometry. In some experiments fragments of thymus were used for nucleosome determination by ELISA. To evaluate the efficiency of the treatments with the three inhibitors to maintain continuous inhibition upon their respective targets, rats treated according to the protocols above were used 1.0, 4.0, 8.0, or 12.0 h after the dose in the morning of the third day of treatment to determine JAK-2, STAT-3, and IRS-1 tyrosine phosphorylation after AG490 treatment; Akt serine phosphorylation after LY294002 treatment; and IRS-1 protein expression after IRS-1ASO treatment.

Short-term culture of thymocytes
To prepare isolated thymocytes, rats were anesthetized; thymuses were obtained and gently passed through a steel net. Cells were washed with and resuspended in ice-cold RPMI 1640 containing penicillin-streptomycin, L-glutamine, and 0.5% fetal calf serum. Twenty-four groups of 5.0 x 106 cells were placed in 2.0-ml culture dishes and treated with leptin (10–8 M), AG490 (10–6 M) + leptin (10–8 M), LY294002 (10–8 M) + leptin (10–8 M), IRS-1ASO (4.0 nmol) + leptin (10–8 M), or IRS-1ASO (4.0 nmol) alone. Apoptosis was evaluated after 12 h using the ELISA nucleosome method.

Determination of blood leptin levels on a time course after exogenous leptin injection
Forty rats were randomly divided into five groups of eight rats each. The first group received no treatment. The second group was subdivided into two groups of four rats; the animals were anesthetized and received a dose of 400 µl leptin 10–6 M or an equal volume of saline through the cava vein. Blood was collected from the tail vein at 3.0 min. The third, fourth, and fifth groups were also subdivided into groups of four rats and treated with an ip injection of 400 µl leptin 10–6 M or an equal volume of saline, and blood was collected from the tail vein at 1, 6, and 12 h, respectively. The rats were anesthetized before blood collection. Leptin was determined in the samples using a commercially available ELISA kit following the protocol suggested by the manufacturer (Linco Research).

Detection of apoptosis by flow cytometry
The thymocytes, prepared as described above, were tested for apoptosis using the annexin-V technique (31). Ninety-six microliters of a cell suspension at the final concentration of 1.0 x 106 cells/ml were incubated with 1.0 µl FITC-conjugated annexin-V and 2.5 µl propidium iodide, as suggested by the manufacturer. Cells were kept on ice and incubated in the dark for 10 min before diluting the cells to 250 µl with binding buffer and analyzing using a FACScalibur flow cytometry analyzer and CellQuest software (Becton Dickinson, San Jose, CA). A total of 10,000 cells were acquired. Unlabeled cells suspended in PBS were used as a negative control and for the determination of gates to be used in the apoptosis assays. Apoptotic cells were measured in the annexin-V-positive propidium iodide-negative quadrant and divided by the total number of cells in the gated region.

Detection of apoptosis by nucleosome ELISA
Rats were treated by ip injection as described for the chronic protocol with saline, leptin, AG490, IRS-1ASO, and/or IRS-1SO. On the morning of the fourth day, the animals were anesthetized, and the thymus was removed by thoracotomy. Isolated thymocytes were treated as described above and used 12 h after treatment. Cells were lysed and centrifuged, and cytoplasmatic histone-associated DNA fragments were determined in the supernatant by ELISA (32), as suggested by the manufacturer.

Flow cytometry for determination of cell markers
Rats were anesthetized as described earlier and the thymus removed by thoracotomy. Suspensions (1.0 x 106 cells/ml) of thymocytes in PBS/2% fetal bovine serum were obtained using a Potter glass. Samples of 100 µl of the freshly prepared cell suspensions were incubated for 20 min in the dark, at room temperature, with 10 µl of the following panels of antibodies: CD3FITC/CD4RPE/CD8RPE-Cy5 or CD4RPE/CD8RPE-Cy5/ObR (the ObR antibody was sc-8325 from Santa Cruz). Thereafter the cells were washed and incubated with FITC-conjugated secondary antibody. A three-color analysis was made using a FACScalibur, and the CellQuest software (Becton Dickinson) was used for quantitative analysis. In addition a cell volume analysis was used to evaluate the expression of cell markers in maturating cells.

RT-PCR
Total RNA was extracted from isolated thymocytes (~107 cells/animal) and hypothalami of Wistar rats, Lepdb (db/db), and C57BLKS/J mice using TRIzol reagent according to the instructions of the manufacturer. Reverse transcription (RT) was carried out using 1.0 µg total RNA using SuperScript reverse transcriptase (200 U/µl) and oligo (dT) (50 mM) in a 30-µl reaction volume (5 x RT buffer, 10 mM deoxynucleotide triphosphate, and 40 U/µl Rnase-free inhibitor). The RTs involved a 50-min incubation at 42 C and a 15-min incubation at 70 C. The PCR products were submitted to 1.5% agarose gel electrophoresis containing ethidium bromide and visualized by excitation under UV light. Photodocumentation was performed using the Nucleovision system (NucleoTech, San Mateo, CA) and band quantification was performed using the Gel Expert software (NucleoTech). First-strand cDNA was PCR amplified using a primer from the transmembrane region of the ObR sequence (NCBI/NM 012596) opposed with two primers capable of determining the short or the long forms of the receptor. The sequence of the transmembrane region primer was 5'-CAG GGC TGT ATG TCA TTG-3'; the sequence of the primer for the short form was 5'-GTG CCC AGG AAC AAT TCT-3'; and the sequence of the primer for the long form was 5'-CCA GAG AAG TTA GCA CTG-3'. Control amplifications were carried out in the presence of RNA template and polymerase but in the absence of reverse transcriptase. The ß-actin mRNA was amplified in all samples as control for quality and amount of RNA using the primers 5'-CGT AAA GAC CTC TAT TGC CAA-3' and 5'-AGC CAT GCC AAA TGT GTC AT-3', based on the sequence NCBI/NM 031144. Reactions were carried out at 94 C for 2 min, followed by 50 cycles, each consisting of 30 sec at 92 C, 30 sec at 50 C, and 1 min at 72 C, followed by single-cycle extension for 10 min at 72 C. ß-Actin was amplified by a similar protocol except that the reaction was limited to 30 cycles. This method, with minor modifications, has been used previously (33).

Immunohistochemistry
Thymus fragments were obtained from three control rats. Hydrated, 5-µm sections of paraformaldehyde-fixed, paraffin-embedded tissue were stained by the double-staining fluorescence method. Sections were incubated for 30 min with 2% normal rabbit or normal goat sera at room temperature and then exposed for 12 h in a moister chamber at 4 C to the panel of primary antibodies against JAK-2 (1:20)/ObR (1:20), STAT-3 (1:20)/ObR (1:20), or Akt (1:50)/ObR (1:20) followed by incubation with FITC-conjugated and rhodamine-conjugated secondary antibodies. Images were obtained with a laser confocal microscope (LSM510; Zeiss, New York, NY). Secondary antibody specificity was tested in a series of positive and negative control measurements. Complete description of the method has been published elsewhere (25).

Statistical analysis
All numerical results are expressed as the mean ± SEM of the indicated number of experiments. The results of blots are presented as direct comparisons of bands in autoradiographs and quantified by densitometry using the Scion Image software (Scion Corp., Frederick, MD). Data were analyzed by the two-tailed unpaired Student’s t test or repeat-measures ANOVA (one-way or two-way ANOVA) followed by post hoc analysis of significance (Bonferroni test) when appropriate, comparing experimental and control groups. The level of significance was set at P < 0.05.


   Results
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 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Time course of blood leptin levels after exogenous leptin administration
The treatment of rats with a single dose of exogenous leptin promoted a variation of blood levels of the hormone that reached a peak at 3.0 min and returned to basal levels after 12 h (Table 1Go).


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  		TABLE 1. Time course of blood leptin levels after exogenous leptin administration

 
Leptin inhibits thymic apoptosis
The treatment of rats with two daily doses of leptin (10–6 M) promoted a significant reduction of apoptosis of thymic cells as determined by the nucleosome-ELISA detection method (~30% inhibition of apoptosis, P < 0.05) (Fig. 1AGo) and the annexin-V/flow cytometry method (~15% inhibition of apoptosis, P < 0.05) (Fig. 1BGo).


Figure 1
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  		FIG. 1. Leptin inhibits baseline apoptosis in thymus. Apoptosis of thymic cells was evaluated by determination of nucleosome formation by ELISA (A) and determination of annexin-V expression by flow cytometry (B); gates were determined by evaluating thymic cell population in a nonlabeled flow cytometry analysis (B, upper graph). Methods are described in Materials and Methods. In all experiments, n = 6; *, P < 0.05. ASU, Arbitrary scanning units.

 
Leptin activates JAK-2/STAT-3 and IRS-1/Akt signaling in thymus
The acute injection of a single dose of leptin (10–6 M) induced the rapid tyrosine phosphorylation of the intracellular kinase JAK-2 in thymic tissue (Fig. 2AGo, first blot). This effect was detected at 1.0 min and lasted for at least 5.0 min. To evaluate the dose dependency of this phenomenon, rats were acutely treated with a single dose of leptin (10–6, 10–8, or 10–10 M), and the thymus was obtained after 3 min (data not shown). The highest tyrosine phosphorylation of JAK-2 was obtained with the dose of 10–6 M (3.5-fold increase vs. control, P < 0.05), although the doses of 10–8 M (2.8-fold increase vs. control, P < 0.05) and 10–10 M (2.3-fold increase vs. control, P < 0.05) were also able to induce activation of JAK-2. The acute treatment with leptin also promoted the tyrosine phosphorylation of STAT-3 beginning at 1.0 min, reaching a peak at 3.0 min, and lasting for at least 5.0 min (Fig. 2AGo, second blot). To evaluate the ability of leptin to activate the IRS-1/Akt signaling pathway, rats were treated with a single dose of leptin (10–6 M), and after 2.0 (IRS-1) or 5.0 (Akt) min, the thymus was obtained. As depicted in Fig. 2AGo (two blots at the bottom), leptin promoted a significant increase in tyrosine phosphorylation of IRS-1 and (Ser473) phosphorylation of Akt. The magnitude of the leptin stimulus was somewhat smaller than the insulin effect on the same signal transducers.


Figure 2
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  		FIG. 2. Leptin activates signal transduction in thymus. A, Total protein extracts of thymus were used in immunoprecipitation (IP) (using JAK-2, STAT-3, or IRS-1 antibodies) and immunoblot (IB) [using phosphotyrosine (pY) antibodies in the anti-JAK-2, -STAT-3, and -IRS-1 immunoprecipitated samples or anti-phospho-(Ser473) Akt, in nonpreimmunoprecipitated samples] assays to evaluate leptin signal transduction. Rats were anesthetized and acutely treated with leptin (Lep; 10–6 M) or insulin (Ins; 10–6 M). Thymuses were obtained after the elapsed times as depicted in the figure (anti-JAK-2 and anti-STAT-3 experiments) or after 2.0 min for IRS-1 experiments and 5.0 min for Akt experiments. B, Coexpression of ObR with JAK-2, STAT-3, and Akt was determined by double-staining confocal microscopy. C, The expression of the short (ObRshort) and long (ObRlong) forms of the leptin receptor were determined in hypothalami and thymocytes of Wistar rats (W), Lepdb, or C57BLKS/J mice (C57) by RT PCR. The ß-actin expression was used as control. D, Coexpression of ObR with CD4 and CD8; and evaluation of lymphocyte subpopulations (CD4+/CD8+/CD3+) in thymus was performed by flow cytometry. Numbers within the graphs represent the proportion of double-positive cells in each group. Numbers in the right-hand margin represent the proportion of triple-positive cells in each group. In A, n = 4; *, P < 0.05 vs. leptin (–)/insulin (–); in B, the figures are representative of three independent experiments; in C the figures are representative of four independent experiments; in D, figures are representative of six independent experiments. SSC-Height, Side scatter height; FSC-Height, forward scatter height. ASU, Arbitrary scanning units.

 
ObR is coexpressed with JAK-2, STAT-3, and Akt in most thymic cells
To evaluate the distribution and cell-specific expression of the ObR, we used three distinct methods. In Fig. 2BGo, the coexpression of ObR with JAK-2 (upper panels), STAT-3 (middle panels), and Akt (lower panels) is seen by double-staining confocal microscopy in most cells of the paraformaldehyde-fixed thymic sections. In fact, most cells of thymic cortex and medulla stained strongly for all the three antigens tested. In addition, the expressions of the long and short forms of the ObR were evaluated in isolated thymocytes of Wistar rats in parallel with long form defective Lepdb mouse and its strain control, C57BLKS/J. As shown in Fig. 2CGo, the short form of the ObR can be detected in hypothalamus and thymocytes of all strains evaluated, whereas the long form can be detected only in hypothalamus and thymocytes of Wistar rats and C57BLKS/J mice. Moreover, by flow cytometry, we observed that the predominant cell population of the thymus, the CD4+/CD8+ double-positive cells, stained consistently for the ObR (Fig. 2DGo). Also, it was observed the presence of a distinct pattern of expression of the antigens in different cell subsets. Therefore, we reanalyzed the CD3-positive thymocytes now subdivided according to the cell volume, a parameter that reflects cell maturation. As depicted in Fig. 3Go, there is a progressive reduction of relative ObR expression in CD4+/CD8+ double-positive cells during the process of maturation.


Figure 3
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  		FIG. 3. ObR expression reduces during thymocyte maturation. Thymocytes were divided according to cell volume in groups R1-R3 (upper graph, numbers represent the relative amount of cells in each group). CD3-positive cells from each group were evaluated for CD4, CD8, and ObR expression by flow cytometry. In all experiments n = 6. Numbers within the graphs represent the proportion of double-positive cells in each group. Numbers in the right-hand margin represent the proportion of triple-positive cells in each group. SSC-Height, Side scatter-height; FSC-Height, forward scatter height.

 
Inhibition of IRS-1 and Akt but not JAK-2 restores leptin-inhibited thymic apoptosis
To evaluate the participation of the JAK/STAT and IRS-1/Akt signaling pathways in the leptin-induced inhibition of apoptosis in thymus, we used the inhibitor of JAK tyrosine kinase activity, AG490 (34, 35); the inhibitor of IRS-1 expression, IRS-1ASO (27); and the inhibitor of PI 3-kinase activity, LY294002 (36). None of the treatment regimens with the inhibitors promoted modifications in health or behavior of the rats. The effectiveness of AG490 was tested by pretreating rats with 100 µl of a 10–4 M AG490 solution followed, after 30 min, by an injection with leptin. As depicted in Fig. 4AGo, AG490 significantly reduced the leptin-induced tyrosine phosphorylation of JAK-2. To evaluate the efficiency of IRS-1ASO, rats were treated for 3 d with two daily doses of IRS-1ASO (or the sense control, IRS-1SO) (4.0 nmol). On the morning of the fourth day, the thymus was obtained and total protein extracts were used in regular immunoblotting experiments. As shown in Fig. 4BGo, IRS-1ASO, but not IRS-1SO, almost completely abolished the expression of IRS-1 in thymus. Finally, to evaluate the effectiveness of LY294002, rats were pretreated with a dose of LY294002 (400 µl, 5.0 µM) and then treated with leptin (after 30 min). After 5 min the thymus was obtained and used in regular immunoblot experiments to evaluate the (Ser473) phosphorylation of Akt. As shown in Fig. 4CGo, LY294002 significantly inhibited leptin-induced activation of Akt. In addition, all three inhibitors were effective to maintain inhibition on their respective targets during the interval between doses (Fig. 4Go, D–F).


Figure 4
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  		FIG. 4. Effects of signal transduction inhibitors. A, Rats were anesthetized and treated with saline or AG490 (100 µl, 10–4 M). After 30 min the animals received either saline or leptin (Lep; 400 µl, 10–6 M). After 1.0 min, the thymus was obtained and used in immunoprecipitation (IP) assays with anti-JAK-2 antibodies. The immunoprecipitates were separated by SDS-PAGE, transferred to nitrocellulose membranes, and immunoblotted (IB) with antiphosphotyrosine (pY) antibodies. B, Rats were treated during 3 d with IRS-1ASO (two daily doses of 4.0 nmol), and on the morning of the fourth day, the thymus was obtained, and total protein extracts were submitted to separation by SDS-PAGE, transferred to nitrocellulose membranes, and immunoblotted with anti-IRS-1 antibodies. C, Rats were anesthetized and treated with saline or LY294002 (400 µl, 5.0 µM). After 30 min the animals received either saline or leptin (400 µl, 10–6 M). After 5.0 min the thymus was obtained and submitted to protein separation by SDS-PAGE, transfer to nitrocellulose membranes, and immunoblotting (IB) with anti-phospho (Ser473) Akt antibodies. D–F, Rats were treated for 3 d with two daily doses of AG490 (100 µl, 10–4 M), LY294002 (400 µl, 5.0 µM), IRS-1ASO (4.0 nmol), or saline. One, 4, 8, or 12 h after the dose in the morning of the third day, rats were anesthetized and received an acute dose of saline or leptin (400 µl, 10–6 M). After 1.0 (JAK-2 and STAT-3, D), 2.0 (IRS-1, D and F), or 5.0 (Akt, E) min, thymuses were obtained and used in immunoprecipitation and immunoblotting experiments to determine tyrosine phosphorylation of JAK-2, STAT-3, and IRS-1 (D), serine phosphorylation of Akt (E), or the protein amount of IRS-1 (F). In all experiments n = 4, *, P < 0.05 vs. control (Ctr); §, P < 0.05 vs. leptin treated (A and C). ASU, Arbitrary scanning units; SO, sense oligonucleotide; ASO, antisense oligonucleotide.

 
Next, we evaluated the outcomes of the treatment of rats with each signal transduction inhibitor on the effect of leptin to inhibit thymic apoptosis. Figure 5Go (A–C) shows that inhibition of IRS-1 expression by IRS-1ASO and inhibition of PI 3-kinase activity by LY294002 were both able to restore baseline apoptosis levels, as detected by flow cytometry and nucleosome ELISA. The treatment with AG490, however, resulted in no modification of the leptin-induced inhibition of apoptosis in the thymus, as shown by flow cytometry (Fig. 5Go, A and B) and nucleosome detection ELISA (Fig. 5CGo).


Figure 5
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  		FIG. 5. Effect of signal transduction inhibition on leptin-induced apoptosis in thymus of living rats. Apoptosis of thymic cells was evaluated by determination of annexin-V by flow cytometry (A and B) and nucleosome formation by ELISA (C). Methods are described in Materials and Methods. In all experiments n = 6; *, P < 0.05 vs. saline (Sal); §, P < 0.05 vs. leptin (Lep).

 
Finally, using freshly isolated thymocytes, we confirmed the results obtained in living rats observing that inhibition of PI 3-kinase activity by LY294002 and inhibition of IRS-1 expression by IRS-1ASO were both able to suppress the effect of leptin to inhibit apoptosis (Fig. 6Go), whereas the inhibition of JAK-2 activity by AG490 exerted no effect on leptin-induced inhibition of apoptosis (Fig. 6Go).


Figure 6
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  		FIG. 6. Effect of signal transduction inhibition on leptin-induced apoptosis in isolated thymocytes. Apoptosis of isolated thymocytes was evaluated by determination of nucleosome formation by ELISA. Methods are described in Materials and Methods. In all experiments n = 4; *, P < 0.05 vs. saline (Sal); §, P < 0.05 vs. leptin (Lep).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In recent years a series of studies have shown the participation of leptin in the modulation of several immunological functions (2). In particular, the role played by leptin in the control of apoptosis of cells of the immune system has been proposed to contribute to the defective immune response of nutrient-deprived individuals and in the pathogenesis of autoimmune diseases (2, 22, 23). Here we evaluated the participation of JAK-2/STAT-3 and IRS-1/PI 3-kinase/Akt signaling pathways in the modulation of thymic apoptosis by leptin.

To optimize the methods for acute and chronic treatment with exogenous leptin, we performed a time-course evaluation of blood leptin levels after the injection of a single dose of the hormone. For the time point of 3 min, exogenous leptin was injected through the cava vein to reproduce the acute treatment protocol used in this study. For the time points of 1, 6, and 12 h, leptin was delivered by ip injection as in the chronic protocol. The treatment with exogenous leptin promoted a rapid and significant increase of blood leptin levels, which lasted for at least 6 h. Because during the chronic treatment we used two daily doses of the hormone, we believe that the rats were hyperleptinemic during most of the experimental period.

Initially we showed, using two distinct methods, that leptin inhibits baseline thymic apoptosis of young rats by 15–30%. These numbers are in agreement with a previous evaluation of the leptin effect on the rate of apoptosis in thymus (22). We next evaluated the capacity of leptin to induce the activation of JAK-2/STAT-3 and IRS-1/PI 3-kinase/Akt signaling in thymus. Lymphocytes, particularly CD4+ cells, are known to express the long form of the leptin receptor (4). However, no study so far has evaluated the expression of the ObR in thymus and its coexpression with proteins involved in transducing the leptin signal. Here we show that most cells of the thymus, including the majority of the CD4+/CD8+ thymocytes, express the ObR. When we evaluated ObR expression in thymocytes previously separated by cell size, we observed that during the process of maturation, there is a progressive reduction of relative ObR expression. This fact has never been reported before and may have an important implication for the role of leptin in the modulation of immune function during the earliest steps of life. Next, we observed that the ObR colocalizes with JAK-2, STAT-3, and Akt in most of these cells. When the rats were treated with leptin, both JAK-2/STAT-3 and IRS-1/Akt signaling pathways were activated. Thus, we can conclude that, in the thymus of young rats, the ObR is expressed in virtually all cells and may be activated by leptin, generating a signal that is transduced through the classical leptin signaling pathway (JAK/STAT) and at least one alternative pathway (IRS-1/Akt). Because leptin can activate JAK-2/STAT-3 signaling only if the long form of the receptor is present (37), we can conclude that at least a fraction of the ObR detected in thymus is composed of the long form of the receptor. This was further explored by RT-PCR that revealed the expression of both the long and the short forms of the ObR in thymus and hypothalamus of Wistar rats and C57BLKS/J mice but only the short form in the same tissues of Lepdb mice.

To evaluate the participation of each signaling pathway (JAK/STAT and IRS-1/PI 3-kinase/Akt) in the leptin-induced inhibition of thymic apoptosis, we used distinct inhibitors of signal transduction. Initially the effectiveness of the inhibitors was tested by immunoblot. Once the inhibition of JAK signaling, IRS-1 expression, and PI 3-kinase activity was assured, rats were submitted to different treatment protocols to test the participation of each signaling cascade in the control of apoptosis. Using these approaches, we found that the inhibition of JAK signaling did not interfere with the capacity of leptin to inhibit apoptosis. However, inhibition of IRS-1 expression and inhibition of PI 3-kinase activity were both equally effective in abolishing the inhibitory effect of leptin on apoptosis in thymus.

Activation of Akt by an array of different signals has been known for a long time to exert an important effect on the control of apoptosis in different cell types (38). Activated Akt catalyzes the phosphorylation of the Bcl-2 family member Bad, inhibiting its activity and therefore inhibiting apoptosis (39). With respect to the antiapoptotic effects of leptin, the participation of Akt has been reported in neuroblastoma cells (40), liver (41), and prostate cancer cells (42). Conversely, no study has provided undisputed evidence for the requirement of the JAK/STAT signaling pathway in leptin-induced inhibition of apoptosis. In fact, according to Dunn et al. (14), leptin, acting in thymocytes through the JAK-2/STAT-3 pathway, regulates negative feedback of the signal but not thymic apoptosis.

The aim of this study was to evaluate the effect of exogenous leptin on the basal rate of thymic apoptosis during early steps of life. However, because most of the experiments were performed in living animals, we cannot discard the possibility that some of the effects herein described were due to indirect mechanisms. One possible indirect mechanism that could play a role in this scenario is the production of adrenocortical hormones. Glucocorticoids are well known for their role in the induction of T cell apoptosis (43). Because leptin is capable of inhibiting adrenocortical function (44), one could argue that defective glucocorticoid action in thymus of leptin-treated rats could explain the inhibition of apoptosis described here. However, there are at least three points that may oppose this possibility. The first one refers to the fact that glucocorticoid-induced T cell apoptosis, which depends on the mitochondria/Apaf-1/caspase-9 pathway (45, 46), cannot be inhibited by the activation of PI 3-kinase/Akt signaling (47). The second point refers to the fact that previous reports have observed the antiapoptotic effects of leptin in isolated cell systems (4, 48), thus occurring independently of the systemic effects of the hormone on adrenocortical function. Finally, in at least one report, it was shown that exogenous leptin is capable of overcoming the proapoptotic effects of glucocorticoids (48). At this time, we cannot reject the possibility of the interaction of leptin with other mechanisms involved in the control of thymic cellular apoptosis. In the near future, it will be of great interest to evaluate whether leptin is able to modulate any other mechanism involved in the control of thymocyte survival and also determine whether leptin inhibits specific types of programed cell death in thymus, such as death by neglect or negative selection.

Thus, taking together the results of the present study and the data of the literature, we conclude that leptin exerts an antiapoptotic effect in the thymus of young rats. This effect is mediated by an IRS-1/PI 3-kinase signaling cascade and is independent on JAK activation. Because the ObR has no intrinsic tyrosine kinase activity, we suspect that an intracellular kinase, other than JAK family members, acts as an intermediary between the leptin receptor and the docking protein IRS-1.


   Acknowledgments
 
We thank Dr. N. Conran for English grammar editing.


   Footnotes
 
This work was supported by grants from Conselho Nacional de Desenvolvimento Científico e Tecnológico and Fundação de Amparo à Pesquisa do Estado de São Paulo.

Disclosure summary: all authors have nothing to declare.

First Published Online July 27, 2006

Abbreviations: FITC, Fluorescein isothiocyanate isomer 1; RPE, phycoerythrin; IRS, insulin receptor substrate; JAK, Janus kinase; ObR, Ob receptor; PI 3-kinase, phosphatidylinositol 3-kinase; RT, reverse transcription; STAT, signal transducer and activator of transcription.

Received February 22, 2006.

Accepted for publication July 17, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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