Leptin Receptor Protein Research Articles

Leptin Regulation of the Mesoaccumbens Dopamine Pathway

Leptin is an adipose-derived hormone that acts on hypothalamic leptin receptors to regulate energy balance. Leptin receptors are also expressed in extrahypothalamic sites including the ventral tegmental area (VTA), critical to brain reward circuitry. We report that leptin targets DA and GABA neurons of the VTA, inducing phosphorylation of signal-transducer-and-activator-of-transcription-3 (STAT3). Retrograde tracing combined with pSTAT3 immunohistochemistry show leptin-responsive VTA neurons projecting to nucleus accumbens (NAc). Assessing leptin function in the VTA, we showed that ob/ob mice had diminished locomotor response to amphetamine and lacked locomotor sensitization to repeated amphetamine injections, both defects reversed by leptin infusion. Electrically stimulated DA release from NAc shell terminals was markedly reduced in ob/ob slice preparations, and NAc DA levels and TH expression were lower. These data define a role for leptin in mesoaccumbens DA signaling and indicate that the mesoaccumbens DA pathway, critical to integrating motivated behavior, responds to this adipose-derived signal.

The role of leptin in leptin resistance and obesity

Although the presence of hyperleptinemia with leptin resistance and obesity has long been recognized, a causal role of elevated leptin in these biological states remains unclear. This article summarizes some recent work from our laboratory supporting the concept that leptin, in and of itself, promotes leptin resistance and such resistance compounds the metabolic impact of diet-induced obesity. Results from multiple studies demonstrate that (1) chronically elevated central leptin decreases hypothalamic leptin receptor expression and protein levels and impairs leptin signaling; (2) leptin resistance and obesity are associated with reduced leptin receptors and diminished maximal leptin signaling capacity; and (3) leptin resistance confers increased susceptibility to diet-induced obesity. In essence, the augmented leptin accompanying obesity contributes to leptin resistance, and this leptin resistance promotes further obesity, leading to a vicious cycle of escalating metabolic devastation.

Effects of cold acclimation and deacclimation on glycogen metabolism in the liver of obese and lean Zucker rats

The effects of long-term cold exposure (10 °C for 11weeks) and of deacclimation from cold (25 °C for 2 weeks, after 10 °C for 9 weeks) were investigated in obese and lean Zucker rats. Morphological change was observed in the liver during cold acclimation and deacclimation. Hepatic glycogen content decreased during cold adaptation, more markedly in obese rats. However, plasma glucose levels remained unchanged. After deacclimation, the glycogen content increased when compared to the original level, conspicuously in lean rats. Leptin receptor protein was decreased by cold acclimation in lean, but not obese, rats. Cold acclimation and deacclimation had no effect on expression of GLUT2 or PGC1 protein in either strain.

Characterization of changes in leptin and leptin receptors in a rat model of preeclampsia

The purpose of this study was to determine the influence of reduced uteroplacental perfusion pressure on plasma leptin and placental leptin receptor expression in rats that develop hypertension in the third trimester of pregnancy. The ovarian arteries and abdominal aortae of pregnant Sprague-Dawley rats (n=9) were constricted surgically on day 14 of gestation and were matched with sham controls. Systolic blood pressure and weight were measured biweekly. Maternal plasma leptin levels, placental leptin receptor abundance, fetal number, fetal weight, and placental weight were determined. Reductions in perfusion pressure induced a significant decrease in maternal plasma leptin. Maternal systolic blood pressure and leptin receptor protein abundance was increased in the experimental group. Litter size and fetal and placental weight were significantly decreased in response to reduced perfusion pressure. Reduced uteroplacental perfusion pressure reduces litter size, fetal and placental weights, and maternal plasma leptin levels and increases placental expression of leptin receptors.

Circulating Leptin Correlates with Left Ventricular Mass in Morbid (Grade III) Obesity before and after Weight Loss Induced by Bariatric Surgery: A Potential Role for Leptin in Mediating Human Left Ventricular Hypertrophy

Obesity is frequently associated with left ventricular hypertrophy, even when uncomplicated by hypertension or diabetes mellitus. Left ventricular hypertrophy is an important risk factor for congestive heart failure. The objective of this study was to evaluate the relationship between leptin and left ventricular mass in uncomplicated, morbid (grade 3) obesity and the existence of leptin receptors and intracellular signaling proteins in the human heart. Left ventricular mass (LVM) was calculated through electrocardiogram reading in normotensive grade III obese patients (World Health Organization classification) undergoing bariatric surgery [laparoscopic adjustable gastric banding (LAGB)] at baseline and 1 yr later. The control group was composed of healthy lean normotensive subjects. Leptin receptors were detected by PCR and immunocytochemistry in human heart biopsies. This study was performed at university hospitals. Thirty-one grade 3 obese patients and 30 healthy nonobese normotensive, age- and sex-matched control subjects were studied. Obese subjects underwent LAGB to induce weight loss and were evaluated at baseline and after 1 yr. LVM, plasma leptin, glucose, insulin levels, and homeostasis model assessment index were higher in obese than in lean controls (P < 0.01); at univariate regression analysis, LVM correlated with body mass index, leptin, and homeostasis model assessment index; at multiple regression analysis, LVM only correlated with leptin levels (P = 0.001). Obese subjects were reevaluated 1 yr after LAGB, when their body mass index changed from 46.2 +/- 1.24 to 36.6 +/- 1.05 kg/m(2) (P < 0.01); the decrease in LVM correlated only with the decrease in leptin levels (P < 0.01). We demonstrated that long and short isoforms of the leptin receptor and intracellular proteins mediating leptin signaling were expressed in human heart by RT-PCR, immunocytochemistry, or both methods. These data suggest that leptin could contribute to the left ventricular hypertrophy in humans.

Leptin receptors are expressed in coronary arteries, and hyperleptinemia causes significant coronary endothelial dysfunction

Obesity is associated with marked increases in plasma leptin concentration, and hyperleptinemia is an independent risk factor for coronary artery disease. As a result, the purpose of this investigation was to test the following hypotheses: 1) leptin receptors are expressed in coronary endothelial cells; and 2) hyperleptinemia induces coronary endothelial dysfunction. RT-PCR analysis revealed that the leptin receptor gene is expressed in canine coronary arteries and human coronary endothelium. Furthermore, immunocytochemistry demonstrated that the long-form leptin receptor protein (ObRb) is present in human coronary endothelium. The functional effects of leptin were determined using pressurized coronary arterioles (<130 microm) isolated from Wistar rats, Zucker rats, and mongrel dogs. Leptin induced pharmacological vasodilation that was abolished by denudation and the nitric oxide synthase inhibitor N(omega)-nitro-l-arginine methyl ester and was absent in obese Zucker rats. Intracoronary leptin dose-response experiments were conducted in anesthetized dogs. Normal and obese concentrations of leptin (0.1-3.0 microg/min ic) did not significantly change coronary blood flow or myocardial oxygen consumption; however, obese concentrations of leptin significantly attenuated the dilation to graded intracoronary doses of acetylcholine (0.3-30.0 microg/min). Additional experiments were performed in canine coronary rings, and relaxation to acetylcholine (6.25 nmol/l-6.25 micromol/l) was significantly attenuated by obese concentrations of leptin (625 pmol/l) but not by physiological concentrations of leptin (250 pmol/l). The major findings of this investigation were as follows: 1) the ObRb is present in coronary arteries and coupled to pharmacological, nitric oxide-dependent vasodilation; and 2) hyperleptinemia produces significant coronary endothelial dysfunction.

Induction of Leptin Receptor Expression in the Liver by Leptin and Food Deprivation

Leptin resistance is a common feature of obesity and the metabolic syndrome. However, the regulated expression of the leptin receptor (Ob-R) has not been studied in detail. Expression profiling of liver mRNA in leptin-treated wild-type mice revealed a marked increase in leptin receptor mRNA levels, which had not previously been described. This was confirmed by isoform-specific real-time PCR, which showed a >25-fold increase in the mRNAs encoding the short forms (Ob-Ra, Ob-Rc) and a >10-fold increase in the mRNA encoding the long (Ob-Rb) form of the leptin receptor in liver. In parallel, we also observed induction of plasma-soluble leptin receptor (SLR) protein by leptin administration, pair feeding, and short term food restriction. However, induction of SLR by leptin is abolished in mice with selective deletion of Ob-R from liver using Cre-LoxP technology. These data suggest that the liver is a major source of Ob-R mRNA expression under conditions of negative energy balance. Membrane-bound Ob-R is then shed into the circulation as SLR. Our study thus reveals an unexpected role of the liver in modulating total circulating leptin levels and possibly its biological activity.

140 EXPRESSION OF LEPTIN LIGAND AND RECEPTOR AND EFFECT OF EXOGENOUS LEPTIN SUPPLEMENTATION ON IN VITRO DEVELOPMENT OF PORCINE IN VITRO FERTILIZED AND SOMATIC CELL NUCLEAR TRANSFER EMBRYOS

The present study investigated the expression of ligand and receptor for leptin, and the effect of leptin supplementation on preimplantation development of porcine in vitro-fertilized (IVF) and somatic cell nuclear transfer (SCNT) embryos. The IVF embryos were produced using frozen boar semen and SCNT embryos were obtained by nuclear transfer of fetal fibroblasts into enucleated oocytes. In Exp. 1, in vitro-matured porcine oocytes and embryos in 2-, 4-, and 8-cell as well as morula and blastocyst stages derived from IVF or SCNT were immunostained for leptin ligand and receptor with their specific antibodies. The expression of leptin ligand and receptor proteins was detected in oocytes and all stages of IVF and SCNT embryos. The IVF (Exp. 2; n = 635, 630, 633, 635, respectively) or SCNT oocytes (Exp. 3; n = 256, 258, 251, 258, respectively) were cultured in modified North Carolina State University (mNCSU)-23 medium supplemented with various concentrations (1, 10, 100, or 1000 ng/mL) of leptin. For the control group, IVF (n = 635) or SCNT embryos (n = 249) were cultured without leptin supplementation (0 ng/mL leptin). Embryo development and cell number in blatoscysts after differential staining according to a modified staining procedure (Thouas et al. 2000 Reprod. Biomed. Online 3, 25–29) were evaluated. The IVF or SCNT embryos were randomly distributed, and experiments were replicated at least 11 times. The differences in embryo development among experimental groups were analyzed using one-way ANOVA after arcsine transformation (without arcsine transformation for cell number of blastocysts) to maintain homogeneity of variance. Post hoc analyses to identify between-group differences were performed using the LSD test. In SCNT embryos, the cleavage rate was not different in leptin-treated groups (73.8, 77.5, 75.7, or 78.7%, respectively) compared to the control (76.3%). The rate of blastocyst formation (at 166 h after the day of injection of donor cells) in SCNT embryos was significantly increased (P &lt; 0.05) in 1000 ng/mL leptin-supplemented group (20.2%) compared with the control (12.9%) and 1 ng/mL leptin-supplemented (12.5%) groups. Supplementing mNCSU-23 with 1000 ng/mL leptin also significantly increased (P &lt; 0.05) the number of total cells (54.6) and trophectoderm (TE) cells (39.1) in SCNT blastocysts (n = &gt;25) compared with the control [45.1 (total cells) and 31.6 (TE cells)] and 10 ng/mL leptin-supplemented group [44.4 (total cells) and 31.7 (TE cells)]. In IVF embryos, leptin supplementation did not affect pre-implantation embryo development and cell number in blastocysts. In conclusion, the present study demonstrated the expression of leptin ligand and receptor proteins in porcine in vitro matured oocytes, IVF and SCNT embryos, and the embryotropic role of leptin in the SCNT embryo development. This study was supported by the Advanced Backbone IT Technology Development (IMT 2000-C1-1) and the Korean Ministry of Science and Technology (M10310060000-03B4606-00000).

Gene expression of leptin, leptin receptor, prolactin receptor and whey acidic protein in mammary glands of late-pregnant gilts from two breeds

In order to identify genes which are essential for pig mammary gland development, mRNA levels of prolactin receptor (PRL-R), leptin, leptin receptor and whey acidic protein (WAP) were measured in parenchymal tissue of 110-d-pregnant gilts. Thirteen Upton-Meishan (UM) and 14 Large White (LW) pregnant gilts and 5 non-pregnant control gilts (2 LW and 3UM) were used. PRL-R and WAP mRNA levels were higher in pregnant than in non-pregnant gilts (P &lt; 0.05). Leptin mRNA levels were higher in UM than in LW gilts (P &lt; 0.05), but this breed effect was not seen when leptin mRNA levels were corrected for percent fat in parenchyma. Correlations were found between concentrations of IGF-I in plasma and PRL-R (P &lt; 0.01) and WAP (P &lt; 0.05) mRNA levels in UM gilts. Serum prolactin (PRL) was correlated with leptin mRNA levels in the overall (P &lt; 0.05) and LW (P &lt; 0.05) populations of gilts, while estradiol was associated with leptin receptor mRNA in UM gilts (P &lt; 0.05). The mRNA levels of all studied genes were positively correlated with mammary parenchymal and extra parenchymal weights in UM gilts, whereas these variables were only correlated with PRL-R and WAP gene expression in LW gilts. The presence of leptin and leptin receptor mRNA in parenchymal tissue suggests a paracrine role for leptin in mammary tissue of late-pregnant gilts. These results also suggest that the PRL signalling pathway is fully active at the transcriptional level in the mammary gland of gilts at 110 d of pregnancy. Key words: Genetics, pig, mammary glands, Meishan, mRNA

Alimentary leptin receptors are most abundant in the gallbladder

Abstract Introduction: We have demonstrated that leptin deficiency and leptin resistance are associated with abnormal motility in the gallbladder. In addition, we have shown that serum glucose and insulin are inversely correlated with biliary motility and that administration of leptin to deficient mice normalizes gallbladder function. Thus, leptin administration may be therapeutic for biliary dyskinesia. However, no data are available on biliary leptin receptor levels. Therefore, we hypothesized that leptin receptors would be abundant in the gallbladder. Methods: 11 week old female lean control (C57Bl/6J, n = 12), leptin-deficient obese/diabetic (Lepob, n = 8), leptin-resistant obese/diabetic (Lepdb, n = 8) and non-obese, diabetic (NOD/LtJ, n = 8) mice were fasted overnight. The next day alimentary tissues including stomach, duodenum/jejunum, ileum, colon, gallbladder, liver and spleen were removed, and leptin receptor (Ob-Rb, long form) protein levels were determined by ELISA. Data were analyzed by Two-way and One-way ANOVA and by Student-Newman-Keuls for pair-wise multiple comparisons. Results: Leptin receptors were similarly abundant in the gallbladder of each strain (p ∗ . C57BI/6J Lepob Lepdb NOD Leptin receptors 22.6 ± 4.9 23.8 ± 3.5 24.6 ± 4.0 35.5 ± 8.3 ∗ ∗ p Conclusions: These data suggest that leptin receptors 1) are found most commonly in the gallbladder and 2) are increased in the gallbladder of non-obese diabetic mice. We conclude that alimentary leptin receptors are most abundant in the gallbladder and that leptin provides a link among obesity, diabetes, and gallstone formation.

Caloric restriction reverses the deficits in leptin receptor protein and leptin signaling capacity associated with diet-induced obesity: role of leptin in the regulation of hypothalamic long-form leptin receptor expression.

The objectives of this study were to determine if reduced long-form leptin receptor (ObRb) expression in diet-induced obese (DIO) animals is associated with deficits in maximal leptin signaling and, secondly, to establish the effects of short-term caloric restriction (CR) on ObRb expression and function. Groups of DIO and life-long chow-fed (CHOW) F344xBN male rats, aged 6 months, were given an i.c.v. injection containing 2 micro g leptin or artificial cerebrospinal fluid (ACSF) vehicle. Leptin induced a >6-fold increase in STAT3 phosphorylation in CHOW rats, but less than 2-fold increase in DIO. Reduced maximal leptin-stimulated STAT3 phosphorylation in DIO rats was coupled with a decline in both ObRb expression and protein. At this point, subgroups of DIO and CHOW animals underwent CR for 30 days and were then tested for acute leptin responsiveness. CR resulted in a 45 and 85% increase respectively in leptin-stimulated STAT3 phosphorylation in CHOW and DIO animals. Similarly, CR increased ObRb expression and protein in both CHOW and DIO animals. To explore the role of leptin in regulating ObRb expression, we reversibly overexpressed leptin in the hypothalamus and found that ObRb mRNA inversely follows central leptin expression. By enhancing both ObRb expression and signaling capacity, CR may enhance leptin responsiveness in leptin-resistant DIO animals.

Leptin receptor expression in fetal lung increases in late gestation in the baboon: a model for human pregnancy.

Leptin produced by both adipose tissue and the placental trophoblast, has been proposed to regulate numerous aspects of human conceptus development. Although recent animal studies have suggested an additional role for the polypeptide in fetal lung maturation, no evidence has been reported in primates. Therefore, we employed the baboon (Papio sp.), a well-characterized primate model for human pregnancy, to determine the presence and ontogeny of leptin receptor in fetal lung with advancing gestation. Lungs were collected from fetal baboons, early in gestation (days 58-62, n = 4), at mid gestation (days 98-102, n = 4), and late in gestation (days 158-165, n = 4) (term 184 days). mRNA transcripts for leptin (LEP) and both long and short intracellular domain isoforms of the leptin receptor (LEP-R(L) and LEP-R(S)) were assessed by RT-PCR. leptin receptor protein was evaluated by immunoblotting and cell types expressing leptin receptor were identified in late pregnancy by immunohistochemistry. Fetal serum leptin concentrations, determined by RIA, remained relatively unchanged at 5.7 +/- 1.1 ng/ml (mean +/- s.e.m.) in mid pregnancy and 8.4 +/- 3.0 ng/ml in late pregnancy (P > 0.05). Although leptin were detectable in fetal lung, no changes in transcript abundance were apparent with advancing gestation. However, transcripts for both LEP-R(L) and LEP-R(S) receptor isoforms increased several-fold (P < 0.05) in fetal lung between mid and late gestation, while leptin receptor protein was detectable only in late pregnancy. leptin receptor was localized in distal pulmonary epithelial cells, including type II pneumocytes. In conclusion, leptin is present in the fetal baboon and its receptor is enhanced during late gestation in cells responsible for the synthesis of pulmonary surfactant. Collectively, these and past findings may suggest a modulatory role for the polypeptide in pulmonary development and/or may identify leptin receptor as a physiological marker of primate fetal lung maturity.

Leptin promotes vascular remodeling and neointimal growth in mice.

Human obesity is associated with elevated leptin levels and a high risk of death from cardiovascular disease. In the present study, we investigated the effects of leptin on vascular wound healing and arterial lesion growth in mice. Wild-type mice placed on an atherogenic, high-fat diet had elevated (9-fold) leptin levels compared with their counterparts maintained on normal chow, and the former demonstrated significantly enhanced neointimal thickening after carotid artery injury with ferric chloride. The lesions forming in response to injury strongly expressed leptin receptor mRNA and protein. Unexpectedly, the atherogenic diet had no effect on injured vessels from leptin-deficient ob/ob mice despite aggravating obesity, diabetes, and hyperlipidemia in these animals. Daily administration of leptin to ob/ob mice during the 3-week period after injury reversed this phenotype, dramatically increasing neointimal thickness and the severity of luminal stenosis. Exogenous leptin also enhanced lesion growth and increased cellular proliferation in injured arteries from wild-type mice but had no effect on vessels from leptin receptor-deficient db/db mice. Our results raise the possibility that there might be a direct, leptin receptor-mediated link between the hyperleptinemia in human obesity and the increased risk for cardiovascular complications associated with this condition.

Leptin induces growth hormone secretion from peripheral blood mononuclear cells via a protein kinase C- and nitric oxide-dependent mechanism.

Leptin is a key mediator of signals regulating food intake and energy expenditure and exerts potent immunomodulatory effects. We investigated the mechanisms mediating the action of leptin on GH secretion from peripheral blood mononuclear cells (PBMCs). Using immunofluorescence microscopy, we demonstrated a polarized expression pattern of leptin receptor protein on the surface of mononuclear cells and constitutive expression of GH in PBMCs. Leptin exhibited a dose-dependent stimulatory effect on GH secretion by PBMCs and also up-regulated the GH receptor gene expression. We did not observe any additive effects of leptin on GH secretion upon activation of cells with the plant mitogen phytohemagglutinin, unlike leptin, phytohemagglutinin exerted no effect on GH receptor mRNA expression. Leptin led to a nitric oxide (NO) synthase (NOS)-specific, dose-dependent increase in NO production from PBMCs because leptin-induced NO release was blocked by the addition of the NOS inhibitor Nomega-Nitro-l-arginine methyl ester and protein kinase C (PKC) inhibitor calphostin C. This leptin-induced GH secretion was dependent on both PKC and NO activation because the addition of PKC and NOS inhibitors inhibited leptin-induced GH production. Although the addition of sodium nitroprusside, a spontaneous liberator of NO, stimulated GH release from PBMCs, leptin had no additive or synergistic effect on sodium nitroprusside-induced GH production. Together, these findings demonstrate a unique action of leptin on immune cells via its ability to stimulate the GH production by blood mononuclear cells via PKC- and NO-dependent pathways. These data also support a probable role for local immune-derived GH in mediating some of the pleiotropic actions of leptin.

Generation of Soluble Leptin Receptor by Ectodomain Shedding of Membrane-spanning Receptors in Vitro and in Vivo

Leptin is an adipocyte-derived hormone with potent effects on food intake and body weight. Genetically obese rodents with mutations of leptin or leptin receptor develop morbid obesity and diabetes. The receptor for leptin, OB-R, is alternatively spliced to at least five transcripts, encoding receptors designated OB-Ra, -b, -c, -d, and -e. OB-Re does not encode a transmembrane domain and is secreted. In humans, transcripts corresponding to OB-Re have not been discovered. However, soluble leptin receptor does circulate in human plasma and represents the major leptin-binding activity. In this report, we attempted to determine whether the soluble leptin receptor may also be derived from membrane-spanning receptor isoforms by ectodomain shedding. Using stable cell lines expressing both OB-Ra, the most abundant leptin receptor isoform, and OB-Rb, the signaling form of the leptin receptor, we demonstrate that soluble leptin receptor protein can indeed be generated by proteolytic cleavage of these two receptor isoforms in vitro. Experiments using adenoviruses expressing dually tagged OB-Ra or Ob-Rb also demonstrate that soluble leptin receptor may be derived from ectodomain shedding of both receptor isoforms in vivo. Because our earlier and other studies have shown that the soluble receptors modulate the levels as well as activity of leptin, our findings suggest that regulated shedding of the ectodomain of membrane-spanning leptin receptors may represent a novel mechanism of modulating leptin's biological activity.

Maternal glucocorticoid treatment modulates placental leptin and leptin receptor expression and materno-fetal leptin physiology during late pregnancy, and elicits hypertension associated with hyperleptinaemia in the early-growth-retarded adult offspring

Leptin concentrations are increased during late pregnancy, and leptin receptors are expressed in placental and fetal tissues, suggesting a role for leptin in placental and/or fetal growth, or both. In humans, leptin concentrations in adulthood are inversely related to body weight at birth, independent of adult adiposity, and correlate with fasting insulin. Glucocorticoids and insulin regulate leptin secretion. Excessive exposure to glucocorticoids during late fetal development in the rat causes intrauterine growth retardation (IUGR), together with hypertension and hyperinsulinaemia in adulthood. Leptin may have a role in the development of some forms of hypertension. To determine whether IUGR induced by maternal glucocorticoid treatment during the last third of pregnancy in the rat is associated with modulation of either maternal or fetal leptin concentrations, the placental expression of leptin or the short form of the leptin receptor (ObR-S), or combinations thereof, and to evaluate whether hypertension or hyperinsulinaemia in the early-growth-retarded adult progeny of dexamethasone-treated dams is associated with altered leptin concentrations. Dexamethasone was administered to pregnant rats from day 15 to day 21 of gestation via a chronically implanted subcutaneous osmotic minipump. Protein expression of leptin and ObR-S in the placenta at day 21 of pregnancy was measured by western blotting. Plasma leptin and insulin concentrations were determined by radioimmunoassay and ELISA respectively. Systolic hypertension was measured by tail cuff plethysmography. Dexamethasone administration during the last third of pregnancy decreased placental mass and fetal body weight at day 21 of gestation, caused maternal hyperleptinaemia but fetal hypoleptinaemia, and suppressed placental leptin protein expression whilst up-regulating placental protein expression of ObR-S. The male and female offspring of dexamethasone-treated dams were hypertensive from 12 weeks of age. One-year-old offspring of dexamethasone-treated dams exhibited significant hyperleptinaemia compared with age-matched controls, an effect associated with hyperinsulinaemia in the male, but not female, offspring. The rat model of maternal dexamethasone treatment is established as a paradigm of 'programmed' hypertension in man. Our data show modification of placental leptin and leptin receptor protein expression by dexamethasone treatment during the last third of pregnancy. We also show that leptin concentrations are suppressed during fetal life but increased in adulthood in this rat model of programmed hypertension. Our data do not necessarily establish a causal relationship between fetal hypoleptinaemia and impaired fetal growth during early life, or between hyperleptinaemia and hypertension in adulthood. Nevertheless, they suggest that hyperleptinaemia may be a component of the cluster of metabolic abnormalities seen in the insulin resistance syndrome in man. They also suggest that excessive fetal exposure to glucocorticoids could be a common early-life stimulus to the association between hyperinsulinaemia, hypertension and hyperleptinaemia often seen in individuals of low birthweight.

Hypothalamic leptin resistance is associated with impaired leptin signal transduction in aged obese rats.

Leptin contributes to the regulation of both food intake and energy expenditure. We previously demonstrated that the F344xBN rat, a rodent model for late-onset obesity, is leptin-resistant and that leptin signal transduction following peripheral administration of leptin is impaired in these aged, overweight rats. To determine if leptin signal transduction is impaired in response to central administration of leptin and whether reduced hypothalamic leptin receptors may be contributing to the impaired signal transduction, we examined the in vivo dose-response leptin-induced STAT3 activation (phosphorylation and binding activity to the SIE M67 oligonucleotide) in response to i.c.v. administration of leptin along with the level of hypothalamic leptin receptor protein in young and older, late-onset obese rats. The leptin-induced maximum phosphorylation of STAT3 was 41% greater in young compared with older obese rats, but the dose required for half-maximal phosphorylation of STAT3 was similar in both the young (41 ng) and old-obese (47 ng) rats. There were no changes in total STAT3 protein with leptin or age, and leptin did not increase phosphorylation of STAT1. Leptin increased phosphorylation of STAT3 transcription factor binding eight-fold in the young but only four-fold in the aged-obese rats, and leptin receptor protein was 50% greater in the young compared with aged rats. These data indicate that aged-overweight rats demonstrate reduced signal transduction in response to centrally administered leptin that may be the result of the diminished leptin receptor protein observed in the aged-obese rats. The diminished leptin receptors and impaired leptin signal transduction may explain the diminished physiological responses observed following leptin administration in older rats. This impaired leptin signal transduction may be due either to the elevated obesity with age or to age itself, or to both.

Expression and regulation of leptin receptor proteins in afferent and efferent neurons of the vagus nerve.

Leptin, the product of the ob gene, plays a key role in the regulation of food intake via a cross-talk between hypothalamic leptin receptors and neuropeptides that affect feeding behaviour. Recent studies have shown a synergistic interaction between leptin and cholecystokinin (CCK) leading to suppression of food intake, which involves CCK-1 receptors and capsaicin-sensitive vagal fibres. In this study, we have investigated the presence of leptin receptors in afferent and efferent neurons of the vagus nerve. By using reverse transcription-polymerase chain reaction, mRNAs encoding long (Ob-Rb) and short (Ob-Ra) leptin receptor isoforms were detected in the rat nodose ganglion, which contains the cell bodies of the vagal afferent neurons. Western blot analysis confirmed the presence of leptin receptor-immunoreactive proteins in extracts from the vagal trunk. Immunohistochemistry showed the presence of leptin receptors and the leptin-induced transcription factor STAT3 in the cytoplasm of nodose ganglion cells. In cervical vagal segments, levels of leptin receptor protein displayed physiological regulation, with decreased amounts after feeding and increased levels after food restriction. In addition, leptin receptor and STAT3 immunoreactivities were detected in neurons of the nucleus of tractus solitarius (NTS) and the dorsal motor nucleus of the vagus nerve (DMNX) by immunofluorescence histochemistry. Furthermore, direct double-labelling demonstrated colocalization of Ob-Rb and STAT3 immunoreactivities in cholinergic vagal efferent cell bodies of the DMNX. It is speculated that vagal leptin receptors, apart from being activated by adipocyte-derived leptin, may also be influenced by leptin produced by the stomach. This may explain the synergistic action of leptin and CCK on neuronal activity in the NTS and on food intake.

Leptin Receptor Immunoreactivity Is Present in Ascending Serotonergic and Catecholaminergic Neurons of the Rat

Using double-labelling immunohistochemistry we have studied the localisation of leptin receptor proteins including both long and short forms and their possible presence in serotonergic (5-HT) and catecholaminergic neurons in the rat brain. Leptin receptor immunoreactivity was found to be widely distributed in the central nervous system including cortical areas, amygdala, several hypothalamic and thalamic nuclei, the raphe system, pontine nuclei, locus coeruleus, parabrachial nucleus, tractus solitarus and the medullary reticular formation. Serotonergic cell groups were identified by 5-HT immunocytochemistry and classified according to standard nomenclature. High degrees of co-existence of leptin receptor immunoreactivity with serotonin in the raphe system were observed in B1, B5, B6, B7, B8 and B9. In B3 and B2 less than 50% of the 5-HT cells colocalised leptin receptor immunoreactivity. Brainstem and diencephalic (catecholaminergic) neurons were identified by tyrosine hydroxylase immunocytochemistry and classified according to standard nomenclature. Within the periventricular hypothalamic dopaminergic nuclei A14 and A12, the metencephalic noradrenergic A6, A7, A2, A1, and the adrenergic C3, C2 and C1 cell groups, nearly all tyrosine hydroxylase-positive cells colocalised with leptin receptor immunoreactivity. In contrast, co-existence of tyrosine hydroxylase and leptin receptor immunoreactivities in the dopaminergic A13, A11, A10, A9 and A8 cell was practically non-existent. Thus leptin, the adipose tissue-derived ligand of the leptin receptor, may in some brain areas directly influence serotonergic, dopaminergic, adrenergic and noradrenergic inputs to the periventricular and medial hypothalamic nuclei.

Serotonergic Neurons Are Targets for Leptin in the Monkey1

Leptin is a secretory product of adipocytes that has been shown to affect food intake, metabolism, and reproduction. One site of leptin's action is the central nervous system, where the leptin receptor (Ob-R) messenger ribonucleic acid (mRNA) and protein are expressed in discrete areas. In both the rat and monkey, Ob-R mRNA has been localized in the Raphe nuclei of the brainstem. Neurons in the Raphe nuclei are the primary source of serotonin in the brain. Serotonergic pathways influence both feeding and reproduction, and these cells are plausible direct targets for leptin's action. We used double label in situ hybridization and computerized image analysis to determine whether serotonergic neurons in the brainstem of the female pigtailed macaque (Macaca nemestrina) express Ob-R mRNA. We observed that many cells in the Raphe nuclei express serotonin transporter mRNA, a marker of serotonergic cells, and Ob-R mRNA. Based on quantitative analysis, the highest number of cells that express both serotonin transporter and Ob-R mRNAs were found in the caudal dorsal Raphe and median Raphe nuclei; fewer double labeled cells were situated in the caudal linear nucleus and rostral median Raphe, whereas double labeled cells occurred infrequently in the rostral dorsal Raphe. These observations suggest that leptin may act on serotonergic cells to mediate some of its effects on ingestive behavior, metabolism, and reproduction.