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Endocrinology Vol. 148, No. 6 2681-2689
Copyright © 2007 by The Endocrine Society

Hypothalamic Apolipoprotein A-IV Is Regulated by Leptin

Ling Shen, Patrick Tso, Stephen C. Woods, Randall R. Sakai, W. Sean Davidson and Min Liu

Departments of Pathology and Laboratory Medicine (L.S., P.T., W.S.D., M.L.) and Psychiatry (S.C.W., R.R.S.), University of Cincinnati College of Medicine, Cincinnati, Ohio 45237

Address all correspondence and requests for reprints to: Min Liu, Ph.D., Department of Pathology and Laboratory Medicine, University of Cincinnati College of Medicine, Cincinnati, Ohio 45237-0507. E-mail: lium{at}uc.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Apolipoprotein A-IV (apo A-IV) is a satiety factor involved in the control of food intake and body weight. Our previous studies demonstrated that apo A-IV is present in areas of the hypothalamus where leptin acts to influence energy homeostasis. In the present studies, we found that leptin-deficient obese (ob/ob) mice have significantly reduced hypothalamic apo A-IV mRNA levels. Intragastric infusion of a lipid emulsion significantly stimulated hypothalamic apo A-IV gene expression in lean controls but not in ob/ob mice. Daily ip administration of leptin (3 µg/g) for 5 d significantly increased hypothalamic apo A-IV mRNA levels of ob/ob mice relative to pair-fed controls. In addition, centrally administered leptin raised the reduced apo A-IV gene expression induced by fasting. Using immunohistochemistry, we demonstrated that apo A-IV is present in leptin-sensitive phosphorylated signal transducer and activator of transcription 3 (pSTAT3)-positive cells of the arcuate nucleus of the hypothalamus. Knockdown of STAT3 expression by small interfering RNA significantly attenuated the stimulatory effect of leptin on apo A-IV protein expression in cultured primary hypothalamic neurons, implying that the hypothalamic apo A-IV is regulated by leptin, at least partially, via the STAT3 signaling pathway. Third-ventricular (intracerebroventricular) administration of a subthreshold dose of leptin (1 µg) potentiated apo A-IV-induced (subthreshold dose, 0.5 µg) reduction of feeding, indicating the existence of a functional synergistic interaction between leptin and apo A-IV, leading to suppression of food intake.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
APOLIPOPROTEIN (APO) A-IV IS a protein secreted together with triglyceride-rich lipoproteins (packaged as chylomicrons) by the small intestine (1, 2). We recently demonstrated that apo A-IV is also expressed in rat hypothalamus (3). Like the regulation of apo A-IV in the intestine, hypothalamic apo A-IV gene expression is reduced by fasting and restored by lipid refeeding (3), and we have found that brain apo A-IV is involved in the control of food intake (4). When administered intracerebroventricularly (icv), apo A-IV dose-dependently inhibits food intake without eliciting signs of sickness (5, 6). Blocking the action of endogenous brain apo A-IV with a selective antibody induces feeding during the light phase when rodents normally eat little (3, 5). These data collectively indicate that hypothalamic apo A-IV has an important role in the control of food intake.

Obesity is a significant health problem leading to increased risk for diabetes and cardiovascular diseases. One well-established risk factor for becoming obese is food consumption in excess of the caloric need of the body. Compelling evidence suggests that obese animals have blunted satiation, raising the possibility that defective satiation signaling from the central nervous system and/or peripheral tissues may contribute to the etiology of obesity. In the present study, we evaluated the hypothesis that brain apo A-IV contributes to energy homeostasis by interacting with the adiposity hormone leptin. We found that relative to lean control mice, genetically obese (ob/ob) mice that lack leptin have significantly decreased levels of apo A-IV in the hypothalamus, although their intestinal and circulating apo A-IV levels are increased. We also found that leptin administration normalized the reduced levels of apo A-IV in ob/ob mice as well as in fasting rats. Using immunohistochemistry and small interfering RNA (siRNA) technology, we further demonstrated that leptin regulates apo A-IV protein expression, at least partially, via signal transducer and activator of transcription 3 (STAT3) signaling pathway in primary hypothalamic neurons. To determine whether there is a direct physiological connection between apo A-IV and leptin, subthreshold doses of both leptin (1 µg) and apo A-IV (0.5 µg) were coadministered icv in 4-h fasted rats. The combination of apo A-IV and leptin, but not the single treatment of either leptin or apo A-IV, significantly reduced food intake relative to vehicle. These data indicate that leptin interacts synergistically with apo A-IV to reduce food intake.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals and surgical procedures
Male adult Sprague Dawley rats (Harlan, Indianapolis, IN), ob/ob, and lean control (C57BL/6J) mice (Jackson Laboratory, Bar Harbor, ME) were individually housed in a temperature-controlled (21 ± 1 C) vivarium on a 12-h light, 12-h dark cycle (lights on at 0600 h). Laboratory chow (Purina 5001) and water were provided ad libitum (except where noted). All animal procedures were performed in accordance with the Institutional Animal Care and Use Committee of the University of Cincinnati.

For implantation of icv guide cannulas, rats were anesthetized ip with ketamine (80 mg/kg)/xylazine (1.6 mg/kg) and implanted with 22-gauge stainless steel cannulas (Plastics One Inc., Roanoke, VA) aimed at the third cerebral ventricle. Coordinates were 2.2 mm posterior to bregma and 7.4 mm ventral to dura on the midline as described previously (7, 8). Placement of cannulas was confirmed by administration of 10 ng angiotensin II in saline while the animals were water replete. Animals that did not drink at least 5 ml water within 30 min after injection were considered to have failed cannula placement and were not used in the experiments. All animals were handled for 5 min daily starting at least 5 d before the experiment to equalize their arousal levels.

Materials
Recombinant mouse leptin was obtained from Dr. A. F. Parlow, National Hormone and Peptide Program, Torrance, CA. Recombinant rat apo A-IV was expressed in Escherichia coli and purified by HPLC in a bioactive form, as described by Liu et al. (6). Rabbit anti-phosphorylated STAT3 (anti-pSTAT3) monoclonal and rabbit anti-STAT3 polyclonal antibodies were purchased from Cell Signaling Technologies (Danvers, MA). Mouse anti-microtubule-associated protein 2 (anti-MAP2) monoclonal antibody was purchased from Chemicon (Temecula, CA).

Effect of fasting and refeeding in mice
Adult male obese ob/ob and lean control mice were fasted overnight (16 h) and respectively assigned to two groups (n = 10 per group) of equal body weight. One group received saline by gavage (0.5 ml/mouse), and the other received lipid (Intralipid, containing 20% lipid, 0.5 ml/mouse). Four hours later, the animals were killed, and their hypothalami and intestines were dissected, immediately frozen in liquid nitrogen, and stored at –80 C. Trunk blood was collected and centrifuged, and the plasma was stored at –80 C. Separate aliquots of plasma were taken for assays of triglycerides, cholesterol, free fatty acids, glucose, and insulin.

Effect of leptin replacement on apo A-IV mRNA
To determine whether reduced hypothalamic apo A-IV mRNA levels in ob/ob mice are due to leptin deficiency or secondary to their elevated body weight, a separate cohort of male adult ob/ob mice were treated for 5 d with daily ip injections of either vehicle or recombinant mouse leptin at a dose of 3 µg/g mouse body weight (9). Pair feeding was performed in a separate group of vehicle-treated mice by assigning each pair-fed ob/ob mouse to a partner in the leptin-treated group. Food intake, water consumption, and body weight were measured daily. Two hours after the final leptin injection, the mice were killed. The hypothalamus of each mouse was dissected, and hypothalamic apo A-IV mRNA levels were measured by quantitative real-time PCR (Q-PCR). Trunk blood was collected, and plasma parameters were measured.

Fasting and leptin treatment in rats
Short-term (e.g. overnight) fasting significantly decreases leptin levels in the circulation (10, 11), which may be related to the changes in apo A-IV gene expression. To determine whether the reduction of brain apo A-IV gene expression induced by fasting (3) is reversed by leptin, we compared hypothalamic apo A-IV mRNA levels in rats that were ad libitum fed or fasted for 36 h. Half of the fasted animals received icv leptin (3.5 µg/rat), and the other half received vehicle 4 h before being killed (i.e. 32 h into the 36-h fast). Animals were killed at 2200 h, and their hypothalamic apo A-IV mRNA levels were measured by Q-PCR. Trunk blood was collected for the measurement of plasma parameters.

Measurement of plasma concentration of lipids, glucose, and insulin of mice and rats
Plasma triglyceride and cholesterol levels were determined using commercially available kits (catalog item TR213 from Randox Laboratories Ltd., Crumlin, Northern Ireland, UK, and catalog item 2350-400H from Thermo Electron Corp., Melbourne, Australia, respectively). Plasma free fatty acid was measured with a NEFA-C kit (catalog item 994-75409 from Wako Chemicals GmbH, Dusseldorf, Germany). Plasma glucose was determined by a commercially available kit (catalog item 220-32 from Diagnostic Chemicals Ltd., Charlottetown, Canada). Plasma insulin levels were measured by rat/mouse insulin ELISA kit (catalog item EZRMI-13K from LINCO Research, Inc., St. Charles, MO).

Northern blotting for intestinal apo A-IV mRNA measurement
Intestinal apo A-IV mRNA levels were determined by Northern blot analysis. Total RNA was isolated from mouse jejunum using Tri Reagent (Molecular Research Center, Inc., Cincinnati, OH) according to the manufacturer’s suggested protocol. Total RNA (15 µg) was separated on denaturing 1.2% agarose-formaldehyde electrophoresis gel, transferred to a Hybond N+ membrane (Amersham Pharmacia Biotech, Piscataway, NJ), and cross-linked by UV light. The membrane was hybridized with 32P-labeled apo A-IV cDNA probe, which was synthesized from random prime labeling. The blots were stripped and reprobed with 32P-labeled 18S by a similar protocol. The levels of expression of a specific apo A-IV mRNA were quantified using a PhosphoImager (Molecular Dynamics, Amersham Biosciences Corp., Sunnyvale, CA) and normalized to the 18S signal.

Q-PCR for hypothalamic apo A-IV mRNA measurement
Hypothalamic total RNA was isolated with Tri Reagent (Molecular Research Center). First-strand cDNA synthesis was done using 1 µg total RNA following the manufacturer’s instructions (Amersham). Q-PCR was performed in a 25-µl final reaction volume with an iCycler iQ Detection System using iQ SYBR Green Supermix (Bio-Rad, Laboratories Inc., Hercules, CA) as described previously (12). Real-time PCR conditions were as follows: 95 C for 3 min for one cycle followed by 38 cycles of 95 C for 30 sec and 58 C for 30 sec on an iCycler iQ real-time PCR detection system. No other products were amplified because melting curves revealed only one peak in each sample. Threshold cycle readings for each of the unknown samples were then used, and the results were transferred and analyzed in Excel using the {delta}{delta}-CT method (PerkinElmer Applied Biosystems, Foster City, CA) (12). Cyclophilin mRNA levels from each sample were used as internal controls to normalize the mRNA levels. The sequences of the primers for rat and mouse apo A-IV and cyclophilin were determined using Primer Design software (Integrated DNA Technologies, Coralville, IA) and listed in Table 1Go.


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  		TABLE 1. Primer sequences and product size for real-time PCR

 
Assay of plasma apo A-IV by ELISA
Plasma apo A-IV levels were measured by the ELISA antibody sandwich method. Standard plasma samples (100 µl of ~0–35 ng/ml apo A-IV) diluted (1:10,000) with PBS containing 0.05% Tween 20 (PBSTw) and 1% BSA were added in duplicate to the coated 96-well plates by 100 µl goat antiserum against rat apo A-IV, which was diluted (1:300) in 0.1 M citrate buffer (pH 3.5). The plates were incubated overnight at 4 C. Then, 100 µl rabbit polyclonal antibody against rat apo A-IV (1:3000 diluted in PBSTw containing 1% BSA) was added and incubated for 2 h at 37 C. After washing, 100 µl peroxidase-conjugated antirabbit IgG (1:200 diluted in PBSTw containing 1% BSA) was added and incubated for 1 h at 37 C. Color was developed by adding 200 µl OPD Peroxidase Substrate (Sigma Chemical Co., St. Louis, MO). After 30 min in the dark at room temperature, 50 µl 3 M HCl was added to stop the reaction, and the optical density was measured at 492 nm using a microplate reader (BioTek Instruments, Inc., Winooski, VT).

Double-labeling immunohistochemistry
Free-floating sections (30 µm) were cut through the hypothalamic arcuate nucleus (ARC) from brains of perfusion-fixed rats. After washing, the sections were blocked with 5% normal donkey serum in PBS containing 0.3% Triton X-100 for 2 h. Then, sections were incubated with a goat antirat apo A-IV antiserum (1:500 dilution) and rabbit anti-pSTAT3 monoclonal antibody (1:100 dilution) overnight at 4 C. Secondary antibodies were used as appropriate, including 1:500 diluted donkey antigoat (for apo A-IV) and donkey antirabbit (for pSTAT3) Ig conjugated to fluorescein, Alexa Fluor 594, or Alexa Fluor 488 (Molecular Probes, Inc., Eugene, OR), respectively. Confocal imaging was performed using a Zeiss 510 microscope system. Omission of the primary antibodies as well as substituting the primary antibody with apo A-IV preabsorbed serum or pSTAT3 preabsorbed antibody were used to determine the specificity of the antibodies.

Primary hypothalamic neuron cultures
Primary neuronal cells were prepared from Sprague Dawley rat embryos on the 18th day of gestation. Hypothalamic tissues were dissected out from the fetal brains and placed in Hanks’ balanced salt solution without Ca2+ and Mg2+ (Invitrogen, Carlsbad, CA) containing 1 mM sodium pyruvate and 10 mM HEPES. The tissues were triturated by repeated pipetting, and the dispersed tissues were allowed to settle for 3 min. The supernatant was transferred to a fresh tube and centrifuged at 200 x g for 2 min. The pellet was resuspended in a serum-free neurobasal medium supplemented with B-27, 0.5 mM L-glutamine, 100 IU/ml penicillin, 100 mg/ml streptomycin, and 25 µM L-glutamic acid (Invitrogen). Then, cells were plated onto 24-well plates or Lab-Tek (Naperville, IL) coverglass chamber slides at a density of 5 x 104 cells/well, seeded, and cultured in a humidified incubator with 95% air and 5% CO2 at 37 C. After a 3-d incubation, half of the medium was replaced with fresh medium with cytosine arabinofuranoside (Ara-C, 10 µM; Sigma) but without L-glutamic acid, and additional incubation at 37 C was carried out. Under these culture conditions, cultured cells contain less than 5% glial cells, confirmed by staining for glial markers.

siRNA transfection and leptin treatment
Six days after seeding (13), the cultured neurons were transfected with Ambion (Austin, TX) predesigned rat STAT3 siRNA, or Ambion validated negative control (scrambled) siRNA. The sense and antisense sequences used for the STAT3, targeting nucleotides 482–502, were 5'-GGUAUCUUGAGAA-GCCAAUtt-3' (sense) and 5'-AUUGGCUUCUCAAGAUACCtg-3' (antisense). Transient transfection of siRNAs was carried out using Lipofectamine 2000 (Invitrogen), according to the manufacturer’s instructions. Briefly, per well of a 24-well culture plate, 1 µl Lipofectamine 2000 was diluted in 50 µl Opti-MEM medium (Invitrogen) and incubated at room temperature for 5 min. In parallel, the appropriate amount of siRNAs (resulting in a final concentration of 40 nM) was diluted in 50 µl Opti-MEM medium. The two mixtures were combined and incubated for 20 min at room temperature for complex formation. The entire mixture was added to primary neuronal culture medium (described above) without antibiotics.

Eight groups were used in the experiment, including two untransfected groups (groups Va and La) without either Lipofectamine 2000 or siRNA treatment for negative control, two groups (groups Vb and Lb) incubated with Lipofectamine 2000 only, two groups (groups Vc and Lc) incubated with a mixture of Lipofectamine 2000 plus STAT3 siRNA, and two groups (groups Vd and Ld) incubated with mixture of Lipofectamine 2000 plus scrambled siRNA. Cells were incubated with the above mixtures, respectively, at 37 C for 2 h, and then the medium was changed back to growth medium for an additional 46 h incubation. Leptin (100 ng/ml) (14) and vehicle were added into the medium of groups La–Ld and groups Va–Vd, respectively, 6 h before the end of the experiment. Then the neurons were washed with ice-cold PBS and stained for immunocytochemistry or harvested for Western blot analysis.

Determination of cell viability
Cell viability after Lipofectamine 2000 treatment or siRNA transfection was monitored using a CellQuati-MTT Cell Viability Assay Kit (BioAssay System, Hayward, CA) according to the manufacturer’s instructions. Briefly, CellQuati-MTT reagent was dissolved in assay buffer, and 15-µl aliquots were added to 96-well plates containing 85 µl medium per well. After incubation for 4 h at 37 C, the absorbance at 570 nm of solubilized MTT-formazan product was measured with Synergy HT Multi-Detection Microplate Reader (BioTek Instruments, Inc., Winooski, VT).

Western blot analysis
Proteins were extracted from cultured primary hypothalamic cells. Briefly, the neurons were washed with ice-cold PBS, harvested with a cell scraper, and lysed in ice-cold lysis buffer [62.5 mM Tris-HCl (pH 6.8), 2% wt/vol SDS, 10% glycerol, 50 mM dithiothreitol, and protease inhibitor cocktails (Sigma)]. Equal amounts (20 µg) of extracted proteins were separated by 4–15% PAGE, transferred onto nitrocellulose membrane, and incubated with STAT3 (1:1000 dilution) or apo A-IV antibody (1:3000 dilution) overnight at 4 C. The amount of immune complexes was quantitated using an enhanced chemiluminescence detection system (Amersham). The blots were stripped and reincubated with monoclonal antibody against actin (Chemicon; 1:10,000 dilution). The reacted membranes were exposed to x-ray film (imaging film from Kodak Scientific, Rochester, NY). Film density, measured as transmittance, was expressed as volume-adjusted OD. The amount of apo A-IV protein was normalized to the respective individual density values reflecting actin protein level and was expressed as a ratio.

Immunocytochemistry
Hypothalamic neurons were grown on four-well Lab-Tek coverglass chamber slides and fixed with 4% paraformaldehyde for 30 min, blocked, and permeabilized in a 3% normal donkey serum, and 0.1% Triton X-100 in PBS for 20 min. Permeabilized neurons were incubated with primary antibodies [goat antirat apo A-IV antibody (1:600) and mouse monoclonal antibody against MAP2 (1:1500)] for 1 h at room temperature and subsequently incubated with the appropriate secondary antibodies [donkey antigoat antibody (for apo A-IV, 1:400 dilution) conjugated with Alexa Fluor 594 and donkey-antimouse antibody (for MAP2, 1:400 dilution) conjugated with Alexa Fluor 488] for 30 min. Samples were visualized with a Zeiss 510 microscope system. Omission of the primary antibodies as well as substituting the primary antibody with apo A-IV preabsorbed serum or mouse normal IgG were used to determine the specificity of MAP2 antibody.

Feeding experiments with apo A-IV and leptin
A novel cohort of male icv-cannulated rats that passed the angiotensin II test was fasted for 4 h before lights-off (1400 h) and then received two injections on each test day. The injections contained vehicle (PBS for leptin) plus saline (vehicle for apo A-IV), vehicle plus apo A-IV (0.5 µg), leptin (1 µg) plus saline, and leptin (1 µg) plus apo A-IV (0.5 µg). The doses of apo A-IV and leptin were subthreshold, having been reported to have no significant effect on food intake or body weight of rats (7, 15). The first icv injection contained either leptin or its vehicle and occurred at 1330 h; it was followed 30 min later by the second icv infusion containing either saline or apo A-IV. Food was then returned, and intake was measured after 30 min and 1, 2, 4, and 24 h. Body weight was recorded after 24 h. Water was available at all times. Each rat received each of the four treatments in random order with at least 5 d occurring between tests for any subject.

Statistical analyses
All values are expressed as mean ± SEM. Data were analyzed using parametric statistics (SigmaStat version 3.1). For comparing apo A-IV gene mRNA or protein levels, one-way or two-way ANOVA followed by Tukey’s test was used. Data from the feeding study were analyzed with two-way repeated-measures ANOVA followed by Tukey’s test. A P value ≤ 0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The ob/ob mice have significantly elevated peripheral, but lower hypothalamic apo A-IV levels
At 8 wk of age, male ob/ob mice were more than 85% heavier than the lean controls (36.7 ± 0.71 vs. 19.5 ± 0.41 g, P < 0.01). Plasma levels of triglyceride, cholesterol, glucose, and insulin were significantly higher in these ob/ob mice compared with lean controls (Table 2Go). Intestinal apo A-IV mRNA and plasma apo A-IV protein levels in ob/ob mice were significantly higher than those in lean mice as determined by Northern blot and ELISA, respectively (P < 0.05, Fig. 1Go). Intraduodenal lipid infusion caused increased plasma triglyceride levels in both ob/ob and lean mice. After lipid administration, significant increases in the intestinal apo A-IV mRNA and plasma apo A-IV protein levels occurred in lean control mice but not in ob/ob mice (Fig. 1Go).


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  		TABLE 2. Plasma concentration of lipid, glucose, and insulin in animals

 

Figure 1
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  		FIG. 1. Comparison of intestinal apo A-IV mRNA (A) and plasma apo A-IV protein (B) levels between ob/ob and lean control (C57BL/6J) mice before and after duodenal infusion of lipid, as determined by Northern blot analyses and ELISA, respectively. Expression of apo A-IV mRNA was normalized to 18 S rRNA. Results are expressed as mean ± SEM; n = 9–10 per group. *, P < 0.05; **, P < 0.01, compared with lean control mice.

 
Unlike apo A-IV levels in peripheral tissue and the circulation, apo A-IV mRNA expression in ob/ob mouse hypothalamus, as measured by Q-PCR, was significantly lower compared with lean control animals (P < 0.05, Fig. 2Go). Intraduodenal lipid infusion to fasted mice significantly increased hypothalamic apo A-IV mRNA content in lean controls but not in ob/ob mice (P < 0.05, Fig. 2Go).


Figure 2
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  		FIG. 2. Comparison of hypothalamic apo A-IV mRNA levels between ob/ob and lean control (C57BL/6J) mice before and after duodenal infusion of lipid, as determined by Q-PCR. Expression of apo A-IV mRNA was the percentage of mRNA level in fasted C57BL/6J mice. Results are expressed as mean ± SEM; n = 9–10 per group. *, P < 0.05, compared with C57BL/6J mice.

 
Leptin normalizes hypothalamic apo A-IV levels in ob/ob mice
To determine whether reduced hypothalamic apo A-IV mRNA levels in the ob/ob mice are due to leptin deficiency or secondary to their elevated body weight, male ob/ob mice were treated with daily ip injections of either vehicle or recombinant mouse leptin (3 µg/g) for 5 d (9). The ob/ob mice receiving leptin reduced their food intake and lost body weight progressively over days. Over the entire experiment, leptin-treated ob/ob mice had significantly decreased food intake (3.2 ± 0.22 g vs. 6.7 ± 0.30 g per day), body weight (6.4% reduction, P < 0.05), and glucose and insulin levels in the plasma (Table 2Go). More importantly, leptin treatment resulted in a significant increase of hypothalamic apo A-IV mRNA levels (0.93 ± 0.54 vs. 0.69 ± 0.45, P < 0.05), compared with vehicle-treated ob/ob mice. The pair-fed mice had a comparable degree of weight loss (5.9% reduction) and similar changes of plasma parameters (Table 2Go), but this intervention did not change hypothalamic apo A-IV mRNA levels (0.73 ± 0.51) (Fig. 3Go), indicating that regulation by leptin of the apo A-IV gene expression is independent of changes in food intake, body weight, or plasma parameters.


Figure 3
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  		FIG. 3. Changes in hypothalamic apo A-IV mRNA levels determined by Q-PCR in ob/ob mice, which had been administered mouse recombinant leptin (3 µg/g) or vehicle daily for 5 d. Values are expressed as mean ± SEM; n = 8 per group. *, P < 0.05, compared with C57BL/6J mice; #, P < 0.05, compared with vehicle-treated ob/ob mice.

 
Leptin normalizes hypothalamic apo A-IV gene expression induced by fasting
To examine whether the previously reported reduction of hypothalamic apo A-IV mRNA induced by fasting (3) can be reversed by leptin, we measured hypothalamic apo A-IV mRNA levels by Q-PCR in rats that were ad libitum fed and fasted for 32 h followed by 4 h of icv leptin (3.5 µg) or vehicle, respectively (total fasting for 36 h). Fasted rats lost a significant amount of body weight (from 273.4 ± 12.4 to 241.2 + 11.8 g) and had significantly reduced hypothalamic apo A-IV mRNA levels, compared with ad libitum-fed controls (0.55 ± 0.08 vs. 1.0 ± 0.10; P < 0.05) (Fig. 4Go). As seen in Table 2Go, fasting significantly reduced plasma triglyceride, glucose, and insulin levels and increased free fatty acid levels but did not affect cholesterol levels. Apo A-IV mRNA expression caused by fasting was significantly increased 4 h after icv leptin (from 0.55 ± 0.08 to 0.87 ± 0.07) (Fig. 4Go). Because no significant difference was found between the groups in the plasma parameters measured (Table 2Go), leptin’s regulation of apo A-IV gene expression cannot be attributed to changes in plasma lipids, glucose, or insulin.


Figure 4
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  		FIG. 4. Leptin reverses the reduction of hypothalamic apo A-IV mRNA levels induced by 36 h fasting. In the fasting plus leptin group, the rats were fasted for 32 h, followed by icv leptin administration (3.5 µg) at the beginning of dark, and the rats were killed 4 h later. Values are expressed as mean ± SEM; n = 7 per group. **, P < 0.05, compared with ad libitum-fed rats; #, P < 0.05, compared with fasted rats.

 
Apo A-IV is colocalized with pSTAT3 in ARC neurons
Leptin long-form receptor (Ob-Rb) is highly expressed in the ARC of the hypothalamus (16). The binding of leptin with Ob-Rb results in phosphorylation of STAT3 and subsequent gene transcription. Thus, the immunohistochemical detection of pSTAT3 expression has served as a useful surrogate for the activation of Ob-Rb (17, 18). To assess whether apo A-IV is present in pSTAT3-positive cells, 3.5 µg leptin or vehicle was administered icv to ad libitum-fed rats (19, 20). Forty minutes later, the rats were perfused via cardiac puncture with ice-cold saline, followed by 4% paraformaldehyde in 0.1 M PBS. The time of 40 min after icv leptin was selected because a previous report indicated that leptin elicits a peak increase of pSTAT3 between 30–45 min (20). Rat brains were removed and postfixed in the same fixative solution at 4 C for 4 h and then submerged in 30% sucrose in 0.1 M PBS at 4 C. We performed confocal microscopic analysis with anti-apo A-IV and anti-pSTAT3 antibodies to characterize apo A-IV in the ARC of leptin- or vehicle-treated rats. Double-labeling immunohistochemistry revealed that apo A-IV is colocalized with pSTAT3 in the cells of ARC (indicated by arrows, Fig. 5Go). Only a few pSTAT3-positive cells were observed in saline control rats (picture not shown). These data provide an anatomical basis for the interaction of leptin with apo A-IV in the regulation of feeding.


Figure 5
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  		FIG. 5. Colocalization of apo A-IV and pSTAT3 in the cells of the ARC. A, Low-magnification view. Rat brains were removed 40 min after leptin or vehicle administration, and the sections were stained with antibodies against apo A-IV (red) and pSTAT3 (green). The colocalization of apo A-IV with pSTAT3 proteins is indicated by arrows. B, High-magnification confocal three-dimensional reconstruction of the area boxed in A, depicting colocalization of apo A-IV and pSTAT3 immunofluorescence. Top, x-z plane; right, y-z plane. Sections are representative of four animals in which staining was examined. 3V, Third ventricle.

 
Apo A-IV is present in primary hypothalamic neurons
Our previous studies demonstrated that apo A-IV is present in the hypothalamus (3, 21), but it is not clear whether apo A-IV exists in neurons or glia. Using dual-labeling immunocytochemistry, we found that apo A-IV is present in cultured primary hypothalamic neurons because it is colocalized with MAP2, a neuronal marker (Fig. 6Go). No immunostaining was observed with apo A-IV preabsorbed control serum (pictures not shown).


Figure 6
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  		FIG. 6. Colocalization of apo A-IV and MAP-2 in primary cultured hypothalamic cells. The cultured cells were stained with antibodies against apo A-IV (red) and MAP-2 (green). The colocalization of apo A-IV with pSTAT3 is reflected as an orange color. Sections are representative of three different cultured cells in which staining was examined.

 
STAT3 knockdown by siRNA significantly attenuates leptin’s stimulatory effect on apo A-IV protein expression in cultured primary hypothalamic neurons
STAT3 is one of the major intracellular mediators of leptin signaling in the hypothalamus (22). To determine whether the STAT3 signaling pathway participates in leptin’s stimulatory effect on apo A-IV, we knocked down STAT3 expression using STAT3 siRNA. We first assessed the effectiveness of siRNA on endogenous expression of STAT3 by exposing cultured hypothalamic neurons to siRNA specific for the STAT3 gene. After a series of pilot studies to optimize the dose-response and time-effect relationships, we found that treatment with STAT3 siRNA, but not scrambled siRNA, at a concentration of 40 nM was effective in knocking down levels of the corresponding protein (STAT3) in the primary hypothalamic neurons as revealed by immunocytochemistry. As depicted in Fig. 7AGo, STAT3-positive cells (red) were significantly reduced after STAT3 siRNA transfection. These results were confirmed by Western blotting (Fig. 7BGo). Compared with controls (Lipofectamine 2000-treated neurons), anti-STAT3 siRNA reduced STAT3 expression by 51% (P < 0.05, Fig. 7CGo). Although the intensity of the bands also slightly decreased in scrambled siRNA-treated neurons, the difference was not significant (P > 0.05, Fig. 7CGo). The cell viability test confirmed that the cells in cultures treated with Lipofectamine 2000 only or Lipofectamine 2000 plus siRNAs were qualitatively indistinguishable from untransfected cells (data not shown), indicating that cytotoxic effects of either Lipofectamine 2000 or siRNAs were not significant. Therefore, we conclude that the effect of the anti-STAT3 siRNA targeting was sequence specific.


Figure 7
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  		FIG. 7. Knockdown of STAT3 by siRNA in cultured primary hypothalamic neurons. A, Fluorescence microscopy revealed that STAT3 expression was inhibited in cultured cells by STAT3 siRNA. B, Representative bands from Western blot: a, Lipofectamine 2000; b, STAT3 siRNA; c, scrambled siRNA; d, untransfected cells. C, Quantification of Western blot. Values are expressed as mean ± SEM; n = 3. **, P < 0.01, compared with Lipofectamine 2000-treated neurons.

 
We then demonstrated that leptin stimulates apo A-IV protein expression in primary hypothalamic neurons. As depicted in Fig. 8Go, leptin-treated untransfected hypothalamic neurons have significantly increased apo A-IV protein levels, compared with levels in vehicle-treated untransfected cells. To determine whether the regulation of apo A-IV expression by leptin requires STAT3 signaling, we measured apo A-IV protein levels by Western blot analysis and found that treatment with STAT3 siRNA slightly reduced apo A-IV basal level (P > 0.05) but significantly attenuated leptin’s stimulatory action on apo A-IV expression (P < 0.05). The reduction in apo A-IV protein expression was 41% in leptin-treated plus STAT3 siRNA-transfected neurons compared with leptin plus Lipofectamine 2000-treated cells (P < 0.05, Fig. 8Go). The control actin expression was not affected by siRNA transfection (Fig. 8Go), indicating the specificity of the STAT3 siRNA effect. These results imply that leptin regulates apo A-IV protein expression, at least partially, via the Janus kinase/STAT3 signaling pathway.


Figure 8
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  		FIG. 8. STAT3 knockdown by siRNA significantly attenuates leptin’s stimulatory effect on apo A-IV protein expression in cultured primary hypothalamic neurons. A, Representative Western blot: a, untransfected cells for control; b, treated with Lipofectamine 2000; c, transfected with STAT3 siRNA; d, transfected with scrambled siRNA. B, Quantification of Western blot. Results are expressed as the percentage of intensity of bands relative to controls. Values are expressed as mean ± SEM; n = 3. *, P < 0.05, compared with vehicle-treated cells; #, P < 0.05, compared with vehicle plus STAT3 siRNA-treated cells.

 
Leptin interacts synergistically with apo A-IV to reduce food intake
As depicted in Fig. 9Go, the icv administration of subthreshold doses of either leptin (1 µg) or apo A-IV (0.5 µg) did not influence food intake, whereas the combination of both leptin (1 µg) and apo A-IV (0.5 µg) significantly reduced food intake measured at 30 and 60 min after the second icv infusion compared with the administration of vehicle plus saline (P < 0.05 in each case). There were no significant differences in food intake after 60 min, and no change of body weight after 24 h, for any group. These data indicate a functional synergism between apo A-IV and leptin in the control of feeding.


Figure 9
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  		FIG. 9. Food intake in response to individual or combined administration of leptin (1 µg) and apo A-IV (0.5 µg) in rats. Values are expressed as mean ± SEM; n = 7. *, P < 0.05, compared with vehicle plus saline control group.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Apo A-IV is an anorectic protein produced in both intestine and hypothalamus, and it has been proposed as a satiety signal (23, 24). Several lines of evidence suggest that brain apo A-IV plays an important role in the control of food intake (3, 5, 6), implying that impairment in the production of hypothalamic apo A-IV could theoretically lead to chronic increases in meal size and the development of obesity. We also hypothesized that the adiposity hormone leptin might interact with the apo A-IV system by regulating its expression. Consistent with this hypothesis, it has been reported that the iv administration of leptin decreases intestinal apo A-IV levels while an animal is actively absorbing fat (25, 26). These findings suggest that peripheral apo A-IV is down-regulated by leptin either directly or secondarily. Therefore, an increased apo A-IV in the intestine and plasma of ob/ob mice with defective leptin signaling (27) was reasonably anticipated and verified in the present study, compared with wild-type C57BL/6J mice. Although the mechanism as to why the obese animals with increased peripheral apo A-IV levels still eat more compared with lean controls is not clear, our data indicate that impaired sensitivity or physiological function occurs in these obese animals.

An unanswered question, however, is whether leptin affects apo A-IV gene expression in the hypothalamus. Because apo A-IV is present in the hypothalamus, where leptin receptors are also expressed, it is possible that leptin may differentially regulate hypothalamic apo A-IV gene expression. Consistent with this possibility, we have demonstrated that these genetically obese mice have significantly decreased apo A-IV mRNA levels in the hypothalamus. This differential regulation of brain and intestinal apo A-IV was also observed when we studied circadian rhythms of apo A-IV levels (12, 28).

Dietary fat is an important factor that is thought to contribute to the development of obesity. In rodents, it has been demonstrated that the concentration of fat in the diet is strongly and positively correlated with the amount of body fat mass, and free access to a high-fat diet leads to obesity (29, 30, 31). We have demonstrated that lipid administration increases apo A-IV gene expression in the hypothalamus (3). If apo A-IV is a physiological regulator of fat intake, a diminished sensitivity to dietary fat-induced apo A-IV activity might contribute to hyperphagia and/or fat preference. Consistent with this hypothesis, we found that ob/ob mice have an attenuated response of apo A-IV production to dietary lipids. The mechanism as to why increased lipid intake did not stimulate the gene expression of the apo A-IV in ob/ob mouse hypothalamus remains obscure. Furthermore, studies are required to determine the mechanisms of why the regulation of peripheral apo A-IV and hypothalamic apo A-IV becomes dysfunctional in obesity as well as the cause and effect between this dysregulation and obesity.

The hormone leptin is primarily synthesized and secreted from adipose tissue and acts in the hypothalamus to decrease food intake and increase energy expenditure (32). Many of these actions of leptin are mediated through the long form of the receptor (Ob-Rb) (33, 34), which is highly expressed in the rat hypothalamus, especially in the ARC (35). In response to leptin, Ob-Rb undergoes a Janus kinase 2-mediated phosphorylation, creating docking sites for cytoplasmic STAT3 molecules (22, 36). Once recruited to the docking sites, STAT3 molecules become phosphorylated, form dimers, and then translocate to the nucleus where they modify gene transcription. STAT3 is therefore one of the major intracellular mediators of leptin signaling in the hypothalamus. Based on this action, hypothalamic leptinoceptive neurons can be visualized by histochemical demonstration of leptin-induced nuclear translocation of the signaling molecule pSTAT3 (20). Taking advantage of this, we used double-labeled immunohistochemistry to reveal that apo A-IV is colocalized with pSTAT3 protein in cells of the ARC after leptin administration. This observation provides anatomical evidence for a direct regulation of leptin on apo A-IV expression in the hypothalamus. Because it has been observed that the phosphorylation of STAT3 protein occurs mainly in neurons (20), the colocalization of apo A-IV with pSTAT3 further supports, but is not conclusive, that apo A-IV is expressed in neurons. Although the mechanism is not clear, leptin may bind to its receptor, resulting in the phosphorylation of STAT3 and a consequent stimulation of apo A-IV gene and protein expression in neurons. Although these results do not establish hypothalamic apo A-IV cells as downstream mediators of leptin action in the control of food intake and energy balance, they strengthen our hypothesis in support of this concept. Consistent with this, conditions associated with either relative (e.g. fasting) or absolute leptin deficiency (e.g. ob/ob obese mice) are associated with reduced apo A-IV mRNA levels in the hypothalamus. Furthermore, leptin treatment for 5 d significantly increases apo A-IV mRNA expression in the hypothalamus of ob/ob mice, whereas pair feeding of vehicle-treated ob/ob mice did not increase apo A-IV mRNA expression despite comparable changes in body weight and plasma parameters, implying that leptin-induced weight loss was not responsible for its ability to increase apo A-IV gene expression in the hypothalamus of ob/ob mice.

In recent years, siRNA, short oligonucleotides of 21–23 nucleotides in length, has provided a powerful experimental tool to enable sequence-specific gene knockdown, allowing efficient analysis of gene function in a multitude of cell types, including mammalian neurons (13, 37). Using this technique, we investigated the mechanism underlying increased apo A-IV expression by leptin and demonstrated that knocking down STAT3 signaling by specific siRNA led to lower apo A-IV expression in cultured hypothalamic neurons. In contrast, no change in the levels of apo A-IV was observed in neurons treated with a negative control (scrambled) siRNA. These data imply that the STAT3 signaling pathway is involved in leptin’s regulation of apo A-IV expression in the hypothalamus.

It has been reported that the effects of leptin in reducing food intake are due to a selective reduction in meal size without changing meal frequency (38, 39), indicating that leptin may exert its anorectic actions through an interaction with meal-related signals involved in the production of satiety. Apo A-IV is one such signal because its production and secretion are meal related and because it plays a role in terminating an individual meal (40). Leptin thus may increase the efficacy of apo A-IV to reduce meal size. To address this, we administered leptin into the brain 30 min preceding the injection of apo A-IV. We found that the combination of subthreshold doses of leptin and apo A-IV elicited a significant reduction in food intake compared with vehicle. No significant anorectic effects were observed when the same doses of leptin and apo A-IV were given separately. These data demonstrate that the functional interaction of leptin and apo A-IV meets the criteria for a synergistic interaction, leading to the significant reduction in food intake (41). Although the mechanisms through which leptin interacts with apo A-IV remain to be determined, leptin-induced modulation of the responsivity of cells within the brain may have increased the efficacy of apo A-IV in the production of cellular activation.

In conclusion, the present results demonstrate that there is decreased apo A-IV gene expression and lower sensitivity of apo A-IV to lipid feeding in the hypothalamus of ob/ob mice. Leptin administration reversed the levels of apo A-IV found in ob/ob mice and in fasted rats. This function may be mediated, at least partially, via the STAT3 signaling pathway. The data from our feeding study indicate that leptin and apo A-IV combine, perhaps synergistically, to reduce food intake. Future studies will be needed to investigate the mechanisms by which obesity and/or increased calorie consumption differentially influence central and peripheral apo A-IV expression in animals and how leptin modulates the responsivity of brain cells to apo A-IV leading to the suppression of food intake.


   Footnotes
 
First Published Online March 15, 2007

Abbreviations: Apo, Apolipoprotein; ARC, arcuate nucleus; icv, intracerebroventricularly; MAP2, microtubule-associated protein 2; Ob-Rb, leptin long-form receptor; PBSTw, PBS containing 0.05% Tween 20; pSTAT3, phosphorylated signal transducer and activator of transcription 3; Q-PCR, quantitative real-time PCR; siRNA, small interfering RNA.

This work was supported by National Institutes of Health Grants DK63907, DK70992, DK56863, DK54504, DK 17844, and DK56910.

Disclosure Statement: The authors have nothing to disclose.

Received November 29, 2006.

Accepted for publication March 8, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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