QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Purchase Article View Shopping Cart Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Jang, M. Articles by Romsos, D. R. Search for Related Content PubMed PubMed Citation Articles by Jang, M. Articles by Romsos, D. R.

---

Articles

Leptin Rapidly Inhibits Hypothalamic Neuropeptide Y Secretion and Stimulates Corticotropin-Releasing Hormone Secretion in Adrenalectomized Mice^{1 ,2}

Miyoung Jang^*, Anahita Mistry^*, Andrew G. Swick and Dale R. Romsos^*3

^* Department of Food Science and Human Nutrition, Michigan State University, East Lansing, MI 48824-1224 and Pfizer Central Research, Groton, CT 06340

³To whom correspondence should be addressed.

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS Effects of ICV leptin... Effects of leptin on... DISCUSSION REFERENCES

 
Leptin may rapidly inhibit food intake by altering the secretion of hypothalamic neuropeptides such as neuropeptide Y (NPY), a stimulator of food intake, and/or corticotropin-releasing hormone (CRH), an inhibitor of food intake. We measured concentrations of NPY and CRH in specific hypothalamic regions [i.e., arcuate nucleus (ARC), paraventricular nucleus (PVN), ventromedial nucleus and dorsomedial nucleus] of 7- to 8-wk-old lean and ob/ob mice at 1 or 3 h after intracerebroventricular leptin administration. No rapid-onset effects of leptin on hypothalamic NPY or CRH concentrations were observed in intact mice. The addition of leptin to hypothalamic preparations from intact mice also did not alter NPY or CRH secretion. Glucocorticoids may oppose leptin actions. Consistent with this, leptin administration to adrenalectomized mice markedly reduced CRH concentrations in the ARC within 3 h after injection. This rapid reduction in CRH concentration in the ARC after leptin administration is more likely due to stimulated CRH release from this region than to decreased synthesis/transport from the PVN because leptin stimulates CRH synthesis in the PVN. Within 20 min after exposure to leptin, NPY secretion from hypothalamic preparations obtained from adrenalectomized mice was lowered by 27% and CRH secretion was elevated by 51%. The current study demonstrates that leptin rapidly influences the secretion of hypothalamic NPY and CRH and that these actions of leptin within the hypothalamus are restrained by the presence of endogenous corticosterone.

KEY WORDS: • neuropeptide Y • corticotropin-releasing hormone • leptin • glucocorticoids • hypothalamus • ob/ob mice

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS Effects of ICV leptin... Effects of leptin on... DISCUSSION REFERENCES

 
Several lines of evidence support the hypothesis that leptin acts within the hypothalamus to influence neuropeptides involved in the regulation of food intake and energy expenditure, including neuropeptide Y (NPY)⁴ and corticotropin-releasing hormone (CRH) (Campfield et al. 1995 , Rohner-Jeanrenaud et al. 1996 , Schwartz et al. 1996 , Stephens et al. 1995 , Wang et al. 1997 ). NPY increases food intake and decreases energy expenditure (Billington et al. 1994 , Clark et al. 1984 ), whereas CRH has opposite actions (Krahn et al. 1988 , Rohner-Jeanrenaud et al. 1989 ). By decreasing the synthesis and/or secretion of hypothalamic NPY and by increasing the synthesis and/or secretion of hypothalamic CRH, leptin would be well positioned to modulate energy balance.

The central administration of leptin, via either a single injection or repetitive injections, decreases NPY mRNA in the ARC of rats and mice (Cusin et al. 1996 , Sahu 1998 , Schwartz et al. 1996 , Stephens et al. 1995 ) and increases CRH mRNA in the PVN of rats (Schwartz et al. 1996 ). These leptin-induced changes in hypothalamic NPY and CRH gene expression have been noted between 6 h and 5 d after leptin administration. Concomitant with the lowering of NPY mRNA, NPY peptide concentrations were also decreased in the arcuate nucleus (ARC), paraventricular nucleus (PVN) and dorsomedial nucleus (DMH) of rats 6 h after the administration of leptin (Wang et al. 1997 ), suggesting less synthesis of NPY. These results provide one possible explanation of how a single injection of leptin might cause relatively long-lasting (i.e., 12–24 h) effects on food intake. Numerous studies, however, demonstrate that the inhibitory actions of leptin on food intake are also rapid in onset, i.e., within 30 min to 2 h (Campfield et al. 1995 , Flynn et al. 1998 , Mistry et al. 1997 , Rentsch et al. 1995 , Seeley et al. 1996 ). These rapid-onset actions of leptin on food intake likely occur via mechanisms other than altered gene expression, given the time typically required for the modulation of protein synthesis. Leptin rapidly, within seconds, depolarizes rat PVN neurons and activates ATP-sensitive potassium channels within the hypothalamus (Glaum et al. 1996 , Powis et al. 1998 , Spanswick et al. 1997 ). These leptin-induced changes in membrane potential and ion channels may lead to rapid changes in neurotransmitter secretion.

Several attempts have been made to examine the acute effects of leptin on hypothalamic NPY and CRH secretion, with inconsistent outcomes. Leptin inhibited NPY release that was first induced by a 2-h exposure of hypothalamic preparations from rats to 0.6 µmol corticosterone/L (Stephens et al. 1995 ). However, in the absence of corticosterone, NPY secretion from this preparation was undetectable. Thus, it was not possible to test the potential effects of leptin on hypothalamic NPY secretion in the absence of added glucocorticoid. The intraperitoneal administration of leptin to rats did not influence in vivo NPY release from the PVN, as measured with a push-pull cannula, during a 2-h time period (Beck et al. 1998 ). The conditions under which leptin might acutely affect NPY secretion remain to be resolved. Likewise, the effects of leptin on CRH secretion are not predictable. Leptin increased CRH secretion, within 20 min, from hypothalamic preparations of rats and mice incubated in 3–5.5 mmol glucose/L (Costa et al. 1997 , Raber et al. 1997 ), but in another report, the addition of leptin to hypothalamic preparations from rats blocked the potentiation of CRH secretion induced by a low concentration (1.1 mmol/L) of glucose (Heiman et al. 1997 ). Glucocorticoids cause rapid-onset changes in the secretion of hypothalamic NPY (stimulatory) and CRH (inhibitory), although the mechanisms are not fully defined (Calogero et al. 1988 , Chen and Romsos 1995 , Stephens et al. 1995 , Suda et al. 1985 ). These actions of glucocorticoids on hypothalamic NPY and CRH secretion are opposite those proposed for leptin. The inhibitory actions of leptin on food intake and body weight are more pronounced in adrenalectomized (ADX) rats and mice than in intact animals (Mistry et al. 1997 , Zakrzewska et al. 1997 ). This observation and the localization of leptin and glucocorticoid receptors within the PVN and ARC (Mercer et al. 1996 , Schwartz et al. 1996 , Tempel and Leibowitz 1994 ), which are important sites for NPY and CRH actions, support the hypothesis that interactions between leptin and glucocorticoids may contribute to the regulation of NPY and CRH secretion (Bray and York 1998 , Flier and Maratos-Flier 1998 ).

The current study was conducted to determine whether leptin administration to mice causes rapid changes in NPY and/or CRH concentrations within specific hypothalamic sites. Changes in hypothalamic NPY and CRH concentrations within a rapid time frame, i.e., within 1–3 h, may in large part reflect changes in transport/secretion rather than in the synthesis of these neuropeptides, because of the time lag typically required for the modulation of gene expression and subsequent protein synthesis. To more directly assess the effects of leptin on the secretion of these neuropeptides, hypothalamic preparations were incubated with and without added leptin. The presence of endogenous leptin and/or glucocorticoids might mask or inhibit the response to administered leptin. Therefore, leptin-deficient ob/ob mice and ADX mice were used in selected studies.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS Effects of ICV leptin... Effects of leptin on... DISCUSSION REFERENCES

 
Animals and diet.

Male lean (ob/+ or +/+) and obese (ob/ob) mice were obtained from our breeding colony of C57BL/6J ob/+ mice. The "Guide for the Care and Use of Laboratory Animals" (National Research Council 1985 ) and local institutional guidelines were followed for the care and treatment of the mice. Mice were weaned at 3–3.5 wk of age, group-housed in solid-bottom plastic cages with wood shavings for bedding and fed a nonpurified diet (Teklad Rodent Diet 8640; Harlan, Bartonville, IL). Room temperature was 23–25°C, and lights were on from 0700 to 1900 h. Mice were studied at 7–8 wk of age.

Reagents.

Coating antisera (rabbit anti-guinea pig IgG for insulin ELISA and goat anti-rabbit IgG for NPY and CRH ELISA) were purchased from EY Laboratories (San Mateo, CA). Guinea pig anti-rat insulin was from Linco Research (St. Charles, MO). Rabbit anti-NPY (human, rat) IgG, rabbit anti-CRH (human, rat) IgG, biotinyl-NPY (human, rat) and biotinyl-CRH were obtained from Peninsula Laboratories (Belmont, CA). NPY (human) and CRH (human, rat) were purchased from Bachem (Torrance, CA). Tissue glue for mounting the hypothalamus was obtained from Miles Inc. (Elkhart, IN). Aprotinin, 2,2'-azino-bis(3-ethylbenz-thiazoline-6-sulfonic acid), ascorbic acid, avidin-peroxidase, bacitracin, HEPES and glucose diagnostic kits were obtained from Sigma Chemical Co. (St. Louis, MO). Bovine serum albumin was from Amresco (Solon, OH). Dextrose was obtained from J. T. Baker Inc. (Phillipsburg, NJ), and the protein assay kit was obtained from BioRad (Hercules, CA). Murine leptin was prepared as previously described (Mistry et al. 1997 ).

Experimental design.

To test the acute actions of leptin on food intake and on hypothalamic NPY and CRH concentrations, lean and ob/ob mice were food deprived for 24 h, injected intracerebroventricularly (ICV) with vehicle or 60 pmol of leptin and refed for 1 h. Some of the vehicle-injected mice were food deprived after the injection to serve as controls for the vehicle-injected refed mice. To determine whether this relatively long period of food deprivation (i.e., 24 h) influenced the ability of exogenous leptin to change hypothalamic NPY and CRH concentrations, other lean and ob/ob mice were not food deprived after the leptin injection to avoid potential confounding effects of leptin-induced differences in food intake or the neuropeptides. The mice were killed 3 h after the leptin injection, rather than 1 h after the leptin injection, as occurred with the former group of mice, to determine whether detectable changes in hypothalamic NPY and CRH concentrations would be evident with a modest increase in the time of exposure to the exogenous leptin (i.e., 3 versus 1 h). Longer lasting effects of leptin on food intake and on hypothalamic NPY and CRH concentrations were determined in non–food-deprived lean and ob/ob mice killed 24 h after the ICV administration of vehicle or 60 pmol leptin. Mice were killed by decapitation between 1500 and 1700 h to avoid circadian variation.

Glucocorticoids might interact with leptin to help regulate body weight (Ur et al. 1996 , Zakrzewska et al. 1997 ). The effects of leptin on hypothalamic NPY and CRH concentrations in ADX lean and ob/ob mice were thus examined. ADX mice were injected ICV with vehicle or leptin (60 pmol) and killed 3 h later (between 1500 and 1700 h). Food was not available during this 3-h period.

Measurements of neuropeptide concentrations in specific hypothalamic nuclei reflect the balance of peptide synthesis and transport, release and degradation rates. To obtain more direct measures of peptide secretion, hypothalamic preparations were incubated in vitro. In vitro hypothalamic secretion of NPY and CRH was compared in lean and ob/ob mice to determine whether the leptin deficiency in ob/ob mice leads to alterations in hypothalamic NPY and CRH secretion. We also examined the effects of leptin on in vitro hypothalamic NPY and CRH secretions in intact and ADX lean and ob/ob mice. Mice were killed between 1300 and 1400 h without food deprivation.

Adrenalectomy.

Mice were adrenalectomized through dorsal incisions while under ether anesthesia at 5 wk of age. Incisions were closed with suture clips. Dexamethasone sodium phosphate (40 µmol/kg body wt) was administered intraperitoneally at surgery to assist in recovery (Feldkircher et al. 1996 ). ADX mice were given free access to food and physiological saline (155 mmol NaCl/L water). Experiments were performed 2 wk after surgery.

ICV injection.

Mice were lightly anesthetized with ether before the ICV injection. Leptin (60 pmol) in 2 µL saline was injected into the lateral ventricle as described previously (Walker and Romsos 1992 ).

Blood sampling.

After the mice were decapitated, trunk blood was collected, and plasma was separated and stored at -20°C for the measurement of glucose and insulin.

Brain sectioning and hypothalamic micropunches.

The brains were quickly removed from the skulls. A cut was made perpendicular to the midline of the mid-hind brain to prepare the tissue for mounting on the specimen holder. The brain was then immediately frozen on dry ice and stored at -80°C. The frozen brain was glued to a specimen holder and placed in a cryostat (Cryocut 1800; Leica, Deerfield, IL) at -10°C. After 30 min, the brain was repeatedly sectioned until the anterior commissure was clearly visible. From this reference point, serial sections of 400–500 µm were sliced according to the stereotaxic atlas of the albino mouse forebrain (Slotnick and Leonard 1975 ). The brain sections were placed on glass slides and kept frozen on dry ice. Discrete hypothalamic nuclei were micropunched under a microscope according to the technique of Palkovits (1973) . Hypothalamic nuclei samples included the ARC, PVN, ventromedial nucleus (VMH) and, in selected trials, DMH (Slotnick and Leonard 1975 ). A 20-gauge, oval-shaped needle was used to micropunch the ARC and PVN, and a round 24-gauge needle was used for the VMH and DMH. These regions were examined because leptin receptors are located within these regions of hypothalamus and are important sites for NPY and CRH action (Frankish et al. 1995 , Krahn et al. 1988 , Tartaglia 1997 ).

Bilateral tissue samples were immediately placed in 100 µL of 0.1 mol HCl/L containing the protease inhibitor aprotinin (90 KIU). The samples were sonicated for 15 s and centrifuged at 10,000 x g for 15 min at 4°C. Supernatants were then lyophilized and stored at -80°C until measurement for NPY and CRH. Tissue pellets were dissolved in 200 µL of 0.1 mol NaOH/L, and protein was determined with a BioRad DC protein kit.

Measurement of hypothalamic NPY and CRH release in vitro.

After rapid removal of the brain from lean and ob/ob mice, the hypothalamus was dissected along the posterior border of the optic chasm, the anterior border of the mammary bodies and the lateral hypothalamic sulci, to a depth of 2 mm. A static incubation system was used to measure neuropeptide release (Kalra et al. 1992 ).

Krebs-Ringer bicarbonate buffer supplemented with 10 mmol HEPES/L, 1 g bovine serum albumin/L, 5.5 mmol glucose/L, 0.3 mmol ascorbic acid/L, 30 mg bacitracin/L and 270,000 KIU aprotinin/L buffer was made fresh and gassed with 95% O₂ and 5% CO₂ for 10 min. The pH was adjusted to 7.4. Dissected hypothalami were immediately placed into polystyrene tubes (12 x 75 mm; Becton Dickinson Labware, Lincoln Park, NJ) containing 750 µL of ice-cold Krebs-Ringer bicarbonate incubation buffer (two hypothalami per tube). Hypothalami were preincubated at 37°C with gentle shaking (30 oscillations/min) for 1 h, and the medium was replaced at 30-min intervals. Hypothalami were then incubated for 1 h (with medium changed at 20-min intervals) for measurements of basal release of NPY and CRH, followed by depolarization with 50 mmol/L to maximize neuropeptide release. The incubation was continued for an additional 20 min with 5 mmol KCl/L to determine whether neuropeptide release induced by 50 mmol KCl/L returned to the basal release level. To test the effects of leptin on the secretion of NPY and CRH, hypothalami were incubated with 30 nmol leptin/L for 40 min (with medium changes at 20-min intervals) after the measurement of basal secretion for 1 h.

Assays.

Plasma glucose was measured with a glucose oxidase-peroxidase kit (Sigma Chemical Co.). Plasma insulin was measured as described by Kekow et al. (1988) with some modifications (Jang and Romsos 1998 ). NPY and CRH were measured with competitive ELISA as described previously (Jang and Romsos 1998 ).

Statistical analysis.

Data are expressed as means ± SEM and were analyzed with SAS/STAT (Version 6.11; SAS Institute, Cary, NC) and SigmaStat (Version 2.0; Jandel Scientific, San Rafael, CA). Differences were considered statistically significant at P < 0.05. Comparisons of hypothalamic concentrations of NPY or CRH in intact mice that were food deprived or refed with or without concurrent leptin administration were examined in a 3 x 2 factorial statistical design. A three-way ANOVA design was used to compare the effects of leptin on hypothalamic concentrations of NPY or CRH in intact or ADX lean and ob/ob mice. A repeated measures one-way ANOVA was used to examine in vitro release of NPY or CRH from hypothalamic preparations. The least significant difference (LSD) test was used for post hoc comparisons. Selected comparisons of lean versus ob/ob mice and intact versus ADX mice were made with the Student’s t test.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS Effects of ICV leptin... Effects of leptin on... DISCUSSION REFERENCES

 
Food intake and plasma insulin and glucose concentrations.

Leptin-treated lean and ob/ob mice consumed 60% less food during a 1-h period, after a 24-h period of food deprivation, than did vehicle-treated mice (0.24 ± 0.03 versus 0.59 ± 0.05 g/h for leptin-treated versus control lean mice, n = 9, and 0.25 ± 0.02 versus 0.64 ± 0.03 g/h for leptin-treated versus control ob/ob mice, n = 8). Lean and ob/ob mice were 7–8 wk old and weighed 23 ± 0.4 g (n = 10) and 37 ± 2 g (n = 10), respectively. Vehicle-treated lean mice (n = 10), as expected, consumed less food in 24 h than did ob/ob mice (n = 8): 3.5 ± 0.2 and 5.4 ± 0.3 g/24 h, respectively. The administration of a single ICV dose of leptin markedly lowered food intake for the next 24 h to 2.2 ± 0.3 g/24 h (-38%) in lean mice (n = 10) and to 1.5 ± 0.3 g/24 h (-72%) in ob/ob mice (n = 8).

Plasma insulin concentrations, as expected, were higher in vehicle-treated ob/ob mice than in their lean counterparts (Table 1 ). Feeding mice for 1 h, after 24 h of food deprivation and the ICV administration of either vehicle or leptin, increased plasma insulin and glucose concentrations (P < 0.05), although plasma insulin concentrations were not increased as much in leptin-treated as in vehicle-treated refed counterparts (P < 0.05) (Table 1) . This effect of leptin on plasma insulin was secondary to a lower food intake induced by leptin, because plasma insulin concentrations in vehicle-treated mice pair-fed to leptin-treated mice also increased less than in mice with free access to food (0.04 ± 0.01 and 0.44 ± 0.1 nmol/L in lean and ob/ob mice, respectively).

	Effects of ICV leptin administration on hypothalamic NPY and CRH concentrations of lean and ob/ob mice

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS Effects of ICV leptin... Effects of leptin on... DISCUSSION REFERENCES

NPY concentrations in the ARC and PVN of ob/ob mice were lower than those in lean mice (Table 2 ). The consumption of food for 1 h lowered NPY concentrations in the PVN of vehicle-treated lean mice (by 36%) but not in ob/ob mice. Leptin administration did not influence the concentrations of NPY in any of the specific hypothalamic regions of these intact lean and ob/ob mice 1 h after refeeding. Leptin administration also did not influence NPY concentrations in any of the specific hypothalamic regions of intact lean and ob/ob mice examined even when food was not provided for a 3-h period to avoid confounding effects of leptin-induced differences in food intake on neuropeptide release (Fig. 1 , left).

View larger version (29K): [in this window] [in a new window]   Figure 1. Effects of intracerebroventricular leptin administration on neuropeptide Y concentrations in the hypothalamus of intact or adrenalectomized (ADX) lean and ob/ob mice. Values are means ± SEM for 6–10 mice. Mice were ADX at 5 wk of age and used at 7 wk of age. Mice were injected intracerebroventricularly with vehicle or leptin (60 pmol) and killed 3 h after the injection, without access to food. ARC, arcuate nucleus; PVN, paraventricular nucleus; VMH, ventromedial nucleus; DMH, dorsomedial nucleus; P, a significant effect of phenotype; A, a significant effect of ADX; L, a significant effects of leptin administration. Comparisons of NPY concentrations were performed with three-way ANOVA. Significant effects were ARC, P, A; PVN, P, A; VMH, P · A; and DMH, A at P < 0.05.

ADX mice had lowered NPY concentrations in the ARC, PVN and VMH of ob/ob mice (35–47% lower) and only in the DMH of lean mice (Fig. 1) . Adrenalectomy elevated CRH concentrations by threefold to fourfold in the ARC of lean and ob/ob mice but lowered CRH concentrations by 48% in the VMH of ob/ob mice (Fig. 2 ). ICV leptin administration to ADX lean mice increased NPY concentrations in the DMH by 70% but did not influence NPY concentrations in any of the other regions examined (Fig. 1) . Leptin administration to ADX lean and ob/ob mice also markedly lowered CRH concentrations by 70–80% within the ARC but did not influence CRH concentrations in the PVN or VMH (Fig. 2) .

View larger version (22K): [in this window] [in a new window]   Figure 2. Effects of intracerebroventricular leptin administration on corticotropin-releasing hormone in hypothalamus of intact or adrenalectomized (ADX) lean and ob/ob mice. Values are means ± SEM for 6–10 mice. Mice were ADX at 5 wk of age and used at 7 wk of age. Mice were injected intracerebroventricularly with vehicle or leptin (60 pmol) and killed 3 h after injection without access to food. ARC, arcuate nucleus; PVN, paraventricular nucleus; VMH, ventromedial nucleus; P, a significant effect of phenotype; A, a significant effect of ADX; L, a significant effects of leptin administration. Comparisons of corticotropin-releasing hormone concentrations were performed by three-way ANOVA. Significant effects were ARC, A, L, P · L, A · L; VMH, P · A at P < 0.05.

Hypothalamus weights did not differ between lean (24 ± 1 mg, n = 10) and ob/ob (23 ± 1 mg, n = 6) mice. Likewise, phenotype did not influence hypothalamic NPY and CRH concentrations: 8 ± 1 and 7 ± 1 pmol NPY and 234 ± 18 and 226 ± 20 fmol CRH, respectively, for lean (n = 7) and ob/ob (n = 5) mice. The release of NPY averaged 11 ± 1 and 10 ± 1 fmol · hypothalamus^-1 · 20 min^-1 from lean (seven incubations) and ob/ob (five incubations) mice, respectively (Fig. 3 ). The release of CRH averaged 17 ± 4 and 13 ± 3 fmol · hypothalamus^-1 · 20 min^-1 from lean and ob/ob mice, respectively (Fig. 3) . The secretion of NPY and CRH in response to 50 mmol KCl/L (to depolarize the neurons) increased onefold to twofold in both lean and ob/ob mice and returned to basal release when the KCl concentration was returned to 5 mmol/L.

View larger version (24K): [in this window] [in a new window]   Figure 3. In vitro release of neuropeptide Y (NPY) and corticotropin-releasing hormone (CRH) from the hypothalamus of lean and ob/ob mice. Data points represent NPY or CRH released per 20-min period per hypothalamus and are given as means ± SEM of five incubations with two hypothalami blocks per chamber. Exposure of the tissue to 50 mmol/L KCl significantly increased NPY or CRH release versus the average basal release, as determined with repeated measures one-way ANOVA (P < 0.05).

	Effects of leptin on in vitro NPY and CRH secretion from the hypothalamus

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS Effects of ICV leptin... Effects of leptin on... DISCUSSION REFERENCES

Leptin (30 nmol/L) did not influence in vitro NPY secretion from the hypothalamus of intact lean mice (7 ± 1 versus 7 ± 1 fmol NPY released · hypothalamus^-1 · 20 min^-1 in the absence and presence of leptin, respectively; seven incubations) or intact ob/ob mice (13 ± 1 versus 12 ± 1 fmol NPY released · hypothalamus^-1 · 20 min^-1 in the absence and presence of 30 nmol/L leptin, respectively; five incubations; Fig. 4 ). Leptin also did not influence in vitro CRH secretion from the hypothalamus of intact lean mice (12 ± 1 versus 11 ± 1 fmol CRH released · hypothalamus^-1 · 20 min^-1 in the absence and presence of 30 nmol/L leptin, respectively; four to six incubations) or ob/ob mice (11 ± 1 versus 10 ± 1 fmol CRH released · hypothalamus^-1 · 20 min^-1 in the absence and presence of 30 nmol leptin/L, respectively; five incubations; Fig. 4 ).

View larger version (24K): [in this window] [in a new window]   Figure 4. Effects of 30 nmol/L leptin on release of neuropeptide Y (NPY) or corticotropin-releasing hormone (CRH) from hypothalamic blocks of lean and ob/ob mice. Data points represent NPY or CRH released per 20-min period and are means ± SEM of four to seven incubations with two hypothalamic blocks per chamber. No significant changes in either NPY or CRH secretion were observed after leptin administration, as determined with repeated measures one-way ANOVA (P < 0.05). Lean and ob/ob mice preparations were obtained on different days, and thus comparisons between lean and ob/ob mice were not made.

ADX significantly lowered hypothalamic NPY (9 ± 1 versus 6 ± 1 pmol in intact and ADX lean mice, respectively; n = 10) and elevated CRH concentrations (389 ± 25 versus 691 ± 63 fmol in intact and ADX mice, respectively; n = 10). ADX did not affect basal secretion of hypothalamic NPY (12 ± 1 versus 11 ± 1 fmol NPY released · hypothalamus^-1 · 20 min^-1 in intact and ADX mice, respectively; five incubations; Fig. 5 ). ADX, as expected (Suda et al. 1985 ), stimulated CRH release by 100%, from 7 ± 0.4 to 16 ± 1 fmol CRH released · hypothalamus^-1 · 20 min^-1 (five incubations; P < 0.05; Fig. 5 ).

View larger version (24K): [in this window] [in a new window]   Figure 5. Effects of 30 nmol/L leptin on in vitro release of neuropeptide Y (NPY) or corticotropin-releasing hormone (CRH) from hypothalamic blocks of intact and adrenalectomized lean mice. Data points represent NPY or CRH released per 20-min period per hypothalamus and are means ± SEM of five incubations with two hypothalamic blocks per chamber. Adrenalectomy significantly elevated basal CRH release without influencing basal NPY secretion as measured with Student’s t test (P < 0.05). There was a significant decrease in NPY release and increase in CRH release after leptin treatment as determined with repeated measures one-way ANOVA (P < 0.05).

Leptin again did not influence CRH secretion from the hypothalamus of intact lean mice (7 ± 0.4 versus 8 ± 1 fmol CRH released · hypothalamus^-1 · 20 min^-1 in the absence and presence of leptin, respectively; five incubations; Fig. 5 ). Leptin administration, however, increased CRH secretion by 51% from the hypothalamus of ADX lean mice (from 16 ± 1 to 25 ± 1 fmol CRH released · hypothalamus^-1 · 20 min^-1; five incubations; P < 0.05; Fig. 5 ).

These leptin actions on NPY and CRH secretion from the hypothalamus of ADX mice occurred rapidly, within 20 min of incubation, and were maintained for the second 20-min period of leptin treatment. These data suggest that leptin and glucocorticoids interact to influence the secretion of hypothalamic NPY and CRH.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS Effects of ICV leptin... Effects of leptin on... DISCUSSION REFERENCES

 
Results from the current study can be summarized as follows. First, leptin administration did not rapidly, i.e., within 1–3 h, influence NPY and CRH concentrations in specific hypothalamic regions of intact mice or in vitro secretion of NPY or CRH from hypothalamic preparations from intact mice. Second, regulation of hypothalamic NPY and CRH secretion was not inherently altered in leptin-deficient ob/ob mice. Third, in the absence of glucocorticoids, leptin rapidly (i.e., within 20 min) decreased NPY secretion and increased CRH secretion.

Mounting evidence suggests that leptin exerts very rapid actions within the hypothalamus, in addition to its slower genomic actions (Banks et al. 1996 , Elmquist et al. 1997 , Powis et al. 1998 , Spanswick et al. 1997 ). We hypothesized that the rapid actions of leptin on food intake in rats and mice were associated with an inhibition of NPY release and/or a stimulation of CRH release within the hypothalamus. Our measurements of changes in NPY and CRH concentrations in specific hypothalamic nuclei within 1–3 h after leptin administration were used as an index of changes in the transport and release of these neuropeptides, with the assumption that minimal effects of leptin on protein synthesis would be evident within this time frame. No rapid-onset effects of leptin on hypothalamic NPY and/or CRH concentrations were observed in intact mice, which is consistent with the ineffectiveness of leptin to alter in vitro NPY and CRH secretions from hypothalamic preparations from these intact mice (Tables 2 and 3 , Figs. 1 , 2 and 4 ).

The possibility that the presence of endogenous leptin in lean mice masked the ability of exogenous leptin treatment to rapidly alter NPY or CRH secretion was tested by administering leptin to leptin-deficient ob/ob mice and by adding leptin to hypothalamic preparations from ob/ob mice. Acute leptin administration did not influence NPY and CRH concentrations within specific hypothalamic nuclei of intact ob/ob mice (Tables 2 and 3 , Figs. 1 and 2 ). Likewise, leptin did not affect the secretion of NPY or CRH from hypothalamic preparations from intact ob/ob mice (Fig. 4) . Longer-term exposure to leptin (i.e., 24 h) did lower NPY concentrations in the ARC, a site of NPY synthesis, of intact ob/ob mice (Table 3) , suggesting a decrease in NPY synthesis in this region secondary to a lowering of NPY mRNA after longer-term exposure to leptin (Sahu 1998 , Schwartz et al. 1996 , Stephens et al. 1995 ). Thus, even though it was possible to detect changes in NPY concentrations within specific hypothalamic nuclei after longer-term (i.e., 24 h) exposure to leptin, we did not obtain any evidence for acute, leptin-induced effects on neuropeptide secretion. Possibly, measurements of NPY and CRH concentrations in specific hypothalamic regions after acute leptin administration were not sensitive enough to detect subtle changes in neuropeptide secretion/transport. Baskin et al. (1999) showed, for example, that not all NPY-containing neurons within the ARC have detectable leptin receptor mRNA. It is also possible that in in vitro measurements, the use of the whole hypothalamus may have masked regional differences in neuropeptide secretion or transport. Alternatively, leptin-induced suppression of food intake in these intact mice may have occurred without direct effects on the hypothalamic release of NPY or CRH.

Glucocorticoids may oppose leptin actions (Ur et al. 1996 , Zakrzewska et al. 1997 ). This suggests that leptin-induced effects on neuropeptide secretion, if evident, would more likely be detected in ADX mice than in intact mice. Indeed, leptin administration to ADX mice markedly reduced ARC CRH concentrations (Fig. 2) . This rapid reduction in CRH concentration (i.e., within 3 h) in the ARC after leptin administration is more likely due to stimulated CRH release from this region rather than to decreased synthesis/transport from the PVN, a site of synthesis, because leptin stimulates CRH synthesis in the PVN (Schwartz et al. 1996 ). Direct evidence for the effects of leptin on neuropeptide secretion was obtained with hypothalamic preparations. Within 20 min after exposure to leptin, NPY secretion from hypothalamic preparations from ADX mice was lowered and CRH secretion was elevated (Fig. 5) . These hypothalamic responses to leptin are consistent with the more dramatic effects of leptin on food intake in ADX rats and mice than in intact animals (Mistry et al. 1997 , Zakrzewska et al. 1997 ).

Our studies were conducted between 1500 and 1700 h, a time of day when corticosterone concentrations are high (Leibowitz 1992 ) and mice are consuming food. Possibly, mice are relatively resistant to leptin at this time. It would be interesting to examine the effects of leptin on NPY and CRH secretion in intact mice at a time of the day when plasma corticosterone concentrations would be low (i.e., shortly after the lights are turned on).

The mechanisms whereby leptin causes rapid changes in NPY and CRH secretion are not yet understood. Leptin has been reported to affect membrane potential and/or ion channels (Powis et al. 1998 , Spanswick et al. 1997 ), cAMP concentrations via stimulation of phosphodiesterase 3B (Zhao et al. 1998 ), nitric oxide generation (Yu et al. 1997 ) and protein kinase C activity (Chen et al. 1997 ). Changes in membrane potential/ion channels, cAMP, nitric oxide and protein kinase C may lead to rapid changes in neurotransmitter secretion. These signal transducers are thus potential candidates to mediate leptin-induced changes in neuropeptide secretion.

The current study demonstrates that leptin exerts rapid-onset actions on the hypothalamic NPY and CRH neuroendocrine system and that these actions of leptin are restrained by the presence of endogenous corticosterone. Other neuronal feeding-regulatory factors, including -melanocyte-stimulating hormone and agouti-related peptides, present within the hypothalamus are also reported to be influenced by leptin (Flier and Maratos-Flier 1998 , Satoh et al. 1998 , Wilson et al. 1999 ). It will be important to consider both the rapid-onset signal transduction actions of leptin and the longer-term effects of leptin on gene transcription to fully understand the role for leptin in the regulation of body energy balance.

	ACKNOWLEDGMENTS

 
We thank Keith Lookingland for his review of the manuscript and Shelli Pfeifer for assistance with manuscript preparation.

	FOOTNOTES

 
¹ Presented at Experimental Biology ’97, New Orleans, LA (Jang, M., Mistry, A., Swick, A. G. & Romsos, D. R. Leptin administration to ob/ob and lean mice failed to change hypothalamic NPY or CRH concentrations); at Experimental Biology ’98, San Francisco, CA (Jang, M. & Romsos, D. R. In vitro hypothalamic secretion of NPY and CRH is not altered in ob/ob mice) and at the 16th International Obesity Conference, Paris, France, 1998 (Jang, M., Mistry, A., Swick, A. G. & Romsos, D. R. ICV administration of leptin markedly decreased corticotropin releasing hormone concentrations in the hypothalamic arcuate nucleus of adrenalectomized ob/ob mice).

² Supported by National Institutes of Health Grant DK-15847.

⁴ Abbreviations used: ADX, adrenalectomized; ARC, arcuate nucleus; CRH, corticotropin-releasing hormone; DMH, dorsomedial nucleus; ICV, intracerebroventricular; NPY, neuropeptide Y; PVN, paraventricular nucleus; VMH, ventromedial nucleus.

Manuscript received March 8, 2000. Initial review completed May 11, 2000. Revision accepted July 11, 2000.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS Effects of ICV leptin... Effects of leptin on... DISCUSSION REFERENCES

 

1. Banks W. A., Kastin A. J., Huang W., Jaspan J. B., Maness L. M. Leptin enters the brain by a saturable system independent of insulin. Peptides 1996;17:305-311[Medline]

2. Baskin D. G., Breininger J. F., Schwartz M. Leptin receptor mRNA identifies a subpopulation of neuropeptide Y neurons activated by fasting in rat hypothalamus. Diabetes 1999;48:828-833[Abstract]

3. Beck B., Kozak R., Stricker-Krongrad A., Burlet C. Neuropeptide Y release in the paraventricular nucleus of Long-Evans rats treated with leptin. Biochem. Biophys. Res. Commun. 1998;242:636-639[Medline]

4. Billington C. J., Briggs J. E., Harker S., Grace M., Levine A. S. Neuropeptide Y in hypothalamic paraventricular nucleus: A center coordinating energy metabolism. Am. J. Physiol. 1994;266:R1765-R1770[Abstract/Free Full Text]

5. Bray G. A., York D. A. The MONALISA hypothesis in the time of leptin. Recent Prog. Horm. Res. 1998;53:95-119

6. Calogero A. E., Gallucci W. T., Gold P. W., Chrousos G. P. Multiple feedback regulatory loops upon rat hypothalamic corticotropin-releasing hormone secretion: potential clinical implications. J. Clin. Invest. 1988;82:767-774

7. Campfield L. A., Smith F. J., Guisez Y., Devos R., Burn P. Recombinant mouse OB protein: Evidence for a peripheral signal linking adiposity and central neural networks. Science 1995;269:546-549(Washington D.C.)[Abstract/Free Full Text]

8. Chen H.-L., Romsos D. R. A single intracerebroventricular injection of dexamethasone elevates food intake and plasma insulin depresses metabolic rates in adrenalectomized obese (ob/ob) mice. J. Nutr. 1995;125:540-545

9. Chen N.-G., Swick A., Romsos D. R. Leptin constrains acetylcholine-induced insulin secretion from pancreatic islets of ob/ob mice. J. Clin. Invest. 1997;100:1174-1179[Medline]

10. Clark J. T., Kalra P. S., Crowley W. R., Kalra S. P. Neuropeptide Y and human pancreatic polypeptide stimulate feeding behavior in rats. Endocrinology 1984;115:427-429[Abstract]

11. Costa A., Poma A., Martignoni E., Nappi G., Ur E., Grossman A. Stimulation of corticotrophin-releasing hormone release by the obese (ob) gene product, leptin, from hypothalamic explants. Neuroreport 1997;8:1131-1134[Medline]

12. Cusin I., Rohner-Jeanrenaud F., Stricker-Krongrad A., Jeanrenaud B. The weight-reducing effect of an intracerebroventricular bolus injection of leptin in genetically obese fa/fa rats. Diabetes 1996;45:1446-1450[Abstract]

13. Elmquist J. K., Ahima R. S., Maratos-Flier E., Flier J. S., Saper C. B. Leptin activates neuron in ventrobasal hypothalamus and brainstem. Endocrinology 1997;138:839-842[Abstract/Free Full Text]

14. Feldkircher K. M., Mistry A. M., Romsos D. R. Adrenalecotmy reverses preexisting obesity in adult genetically obese (ob/ob) mice. Int. J. Obes. 1996;20:232-235

15. Flier J. S., Maratos-Flier E. Obesity and the hypothalamus: Novel peptides for new pathways. Cell 1998;92:437-440[Medline]

16. Flynn M. C., Scott T. R., Pritchard T. C., Plata-Salaman C. R. Mode of action of OB protein (leptin) on feeding. Am. J. Physiol. 1998;275:R174-R179

17. Frankish H. M., Dryden S., Hopkins D., Wang Q., Williams G. Neuropeptide Y, the hypothalamus, and diabetes: insights into the central control of metabolism. Peptides 1995;16:757-771[Medline]

18. Glaum S. R., Hara M., Bindokas V. P., Lee C. C., Polonsky K. S., Bell G. I., Miller R. J. Leptin, the obese gene product, rapidly modulates synaptic transmission in the hypothalamus. Mol. Pharmacol. 1996;50:230-235[Abstract]

19. Heiman M. L., Ahima R. S., Craft L. S., Schoner B., Stephens T. W., Flier J. S. Leptin inhibition of the hypothalamic-pituitary-adrenal axis in response to stress. Endocrinology 1997;138:3859-3863[Abstract/Free Full Text]

20. Jang M., Romsos D. R. Neuropeptide Y and corticotropin-releasing hormone concentrations within specific hypothalamic regions of lean mice, but not ob/ob mice, respond to food-deprivation and refeeding. J. Nutr. 1998;128:2520-2525[Abstract/Free Full Text]

21. Kalra P. S., Phelps C. P., Sahu A., Dube M. G. Quantitation of in vivo and in vitro neuropeptide secretion under the influence of steroids. Neuroprotocols 1992;1:87-97

22. Kekow J., Ulrichs K., Muller-Ruchholtz W., Gross W. L. Measurement of rat insulin: Enzyme-linked immunosorbent assay with increased sensitivity, high accuracy, and greater practicability than established radioimmunoassay. Diabetes 1988;37:321-326[Abstract]

23. Krahn D. D., Gosnell B. A., Levine A. S., Morley J. E. Behavioral effects of corticotropin-releasing factor: Localization and characterization of central effects. Brain Res 1988;443:63-69[Medline]

24. Leibowitz S. F. Neurochemical-neuroendocrine systems in the brain controlling macronutrient intake and metabolism. Trends Neurosci 1992;15:491-497[Medline]

25. Mercer J. G., Hoggard N., Williams L. M., Lawrence C. B., Hannah L. T., Morgan P. J., Trayhurn P. Coexpression of leptin receptor and preproneuropeptide Y mRNA in arcuate nucleus of mouse hypothalamus. J. Neuroendocrinol. 1996;8:733-735[Medline]

26. Mistry A. M., Swick A. G., Romsos D. R. Leptin rapidly lowers food intake and elevates metabolic rates in lean and ob/ob mice. J. Nutr. 1997;127:2065-2072[Abstract/Free Full Text]

27. National Research Council Guide for the Care and Use of Laboratory Animals 1985 National Institutes of Health Bethesda, MD. publication no. 85-23 (rev.)

28. Palkovits M. Isolated removal of hypothalamic or other brain nuclei of the rat. Brain Res 1973;59:449-450[Medline]

29. Powis J. E., Bains J. S., Ferguson A. V. Leptin depolarizes rat hypothalamic paraventricular nucleus neurons. Am. J. Physiol. 1998;274:R1468-R1472[Abstract/Free Full Text]

30. Raber J., Chen S., Mucke L., Feng L. Corticotropin-releasing factor and adrenocorticotrophic hormone as potential central mediators of ob effects. J. Biol. Chem. 1997;272:15057-15060[Abstract/Free Full Text]

31. Rentsch J., Levens N., Chiesi M. Recombinant ob-gene product reduces food intake in fasted mice. Biochem. Biophys. Res. Commun. 1995;214:131-136[Medline]

32. Rohner-Jeanrenaud F., Walker C.-D., Greco-Perotto R., Jeanrenaud B. Central corticotropin-releasing factor administration prevents the excessive body weight gain of genetically obese (fa/fa) rats. Endocrinology 1989;124:733-739[Abstract]

33. Rohner-Jeanrenaud R., Cusin I., Sainsbury A., Zakrzewska K. E., Jeanrenaud B. The loop system between neuropeptide Y and leptin in normal and obese rodents. Horm. Metab. Res. 1996;28:642-648[Medline]

34. Sahu A. Evidence suggesting that galanin (GAL), melanin-concentrating hormone (MCH), neurotensin (NT), proopiomelanocortin (POMC), and neuropeptide Y (NPY) are targets of leptin signaling in the hypothalamus. Endocrinology 1998;139:795-798[Abstract/Free Full Text]

35. Satoh N., Ogawa Y., Katsuura G., Numata Y., Masuzaki H., Yoshimasa Y., Nakao K. Satiety effect and sympathetic activation of leptin are mediated by hypothalamic melanocortin system. Neurosci. Lett. 1998;249:107-110[Medline]

36. Schwartz M. W., Seeley R. J., Campfield L. A., Burn P., Baskin D. G. Identification of targets of leptin action in rat hypothalamus. J. Clin. Invest. 1996;98:1101-1106[Medline]

37. Seeley R. J., Dijk G. V., Campfield L. A., Smith F. J., Burn O., Nelligan J. A., Bell S. M., Baskin D. G., Woods S. C., Schwartz M. W. Intraventricular leptin reduces food intake and body weight of lean rats but not obese Zucker rats. Horm. Metab. Res. 1996;28:664-668[Medline]

38. Slotnick B. M., Leonard C. M. A Stereotaxic Atlas of the Albino Mouse Forebrain 1975 U.S. Department of Health, Education, and Welfare Rockville, MD.

39. Spanswick D., Smith M. A., Groppi V. E., Logan S. D., Ashford M.L.J. Leptin inhibits hypothalamic neurons by activation of ATP-sensitive potassium channels. Nature (Lond.) 1997;390:521-525[Medline]

40. Stephens T. W., Basinski M., Bristow P. K., Bue-Valleskey J. M., Burgett S. G., Craft L., Hale J., Hoffmann J., Hsiung H. M., Kriauciunas A., MacKellar W., Rosteck P. R., Jr, Schoner B., Smith D., Tinsley F. C., Zhang X.-Y., Heiman M. The role of neuropeptide Y in the antiobesity action of the obese gene product. Nature (Lond.) 1995;377:530-532[Medline]

41. Suda T., Yajima F., Tomori N., Demura H., Shizume K. In vitro study of immunoreactive corticotropin-releasing factor release from the rat hypothalamus. Life Sci 1985;37:1499-1505[Medline]

42. Tartaglia L. A. The leptin receptor. J. Biol. Chem. 1997;272:6093-6096[Free Full Text]

43. Tempel D. L., Leibowitz S. F. Adrenal steroid receptors: interactions with brain neuropeptide systems in relation to nutrient intake and metabolism. J. Neuroendocrinol. 1994;6:479-501[Medline]

44. Ur E., Grossman A., Despres J.-P. Obesity results as a consequence of glucocorticoid induced leptin resistance. Horm. Metab. Res. 1996;28:744-747[Medline]

45. Walker H. C., Romsos D. R. Glucocorticoids in the CNS regulate BAT metabolism and plasma insulin in ob/ob mice. Am. J. Physiol. 1992;262:E110-E117[Abstract/Free Full Text]

46. Wang Q., Bing C., Al-Barazanji K., Mossakowaska D. E., Wang X.-M., McBay D. L., Neville W. A., Taddayon M., Pickavance L., Dryden S., Thomas M.E.A., McHale M. T., Gloyer I. S., Wilson S., Buckingham R., Arch J.R.S., Trayhurn P., Williams G. Interactions between leptin and hypothalamic neuropeptide Y neurons in the control of food intake and energy homeostasis in the rat. Diabetes 1997;46:335-341[Abstract]

47. Wilson B. D., Bagnol D., Kaelin C. B., Ollmann M. M., Gantz I., Watson S. J., Barsh G. S. Physiological and anatomical circuitry between agouti-related protein and leptin signaling. Endocrinology 1999;140:2387-2397[Abstract/Free Full Text]

48. Yu W. H., Walczewska A., Karanth S., McCann M. Nitric oxide mediates leptin-induced luteinizing hormone-releasing hormone (LHRH) and LHRH and leptin-induced LH release from the pituitary gland. Endocrinology 1997;138:5055-5058[Abstract/Free Full Text]

49. Zakrzewska K. E., Cusin I., Sainsbury A., Rohner-Jeanrenaud F., Jeanrenaud B. Glucocorticoids as counterregulatory hormones of leptin: toward an understanding of leptin resistance. Diabetes 1997;46:717-719[Abstract]

50. Zhao A. Z., Bornfeldt K. E., Beavo J. A. Leptin inhibits insulin secretion by activation of phosphodiesterase 3B. J. Clin. Invest. 1998;102:869-873[Medline]

This article has been cited by other articles:

				P. Kok, S. W. Kok, M. M. Buijs, J. J. M. Westenberg, F. Roelfsema, M. Frolich, M. P. M. Stokkel, A. E. Meinders, and H. Pijl Enhanced circadian ACTH release in obese premenopausal women: reversal by short-term acipimox treatment Am J Physiol Endocrinol Metab, November 1, 2004; 287(5): E848 - E856. [Abstract] [Full Text] [PDF]

				C. Schulz, K. Paulus, and H. Lehnert Central Nervous and Metabolic Effects of Intranasally Applied Leptin Endocrinology, June 1, 2004; 145(6): 2696 - 2701. [Abstract] [Full Text] [PDF]

				J.-W. Lee and D. R. Romsos Leptin Administration Normalizes Insulin Secretion from Islets of Lepob/Lepob Mice by Food Intake-Dependent and -Independent Mechanisms Experimental Biology and Medicine, February 1, 2003; 228(2): 183 - 187. [Abstract] [Full Text] [PDF]

				A. Sainsbury, C. Schwarzer, M. Couzens, and H. Herzog Y2 Receptor Deletion Attenuates the Type 2 Diabetic Syndrome of ob/ob Mice Diabetes, December 1, 2002; 51(12): 3420 - 3427. [Abstract] [Full Text] [PDF]

				H. Watanobe and S. Habu Leptin Regulates Growth Hormone-Releasing Factor, Somatostatin, and alpha -Melanocyte-Stimulating Hormone But Not Neuropeptide Y Release in Rat Hypothalamus In Vivo: Relation with Growth Hormone Secretion J. Neurosci., July 15, 2002; 22(14): 6265 - 6271. [Abstract] [Full Text] [PDF]

				A. M. Madiehe, L. Lin, C. White, H. D. Braymer, G. A. Bray, and D. A. York Constitutive activation of STAT-3 and downregulation of SOCS-3 expression induced by adrenalectomy Am J Physiol Regulatory Integrative Comp Physiol, December 1, 2001; 281(6): R2048 - R2058. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Purchase Article View Shopping Cart Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal